Method for the detection of the antibiotic resistance spectrum of Mycobacterium species

ABSTRACT

Method for the detection of the antibiotic resistance spectrum of Mycobacterium species present in a sample, possibly coupled to the identification of the Micobacterium species involved, comprising the steps of: (i) if need be releasing, isolating or concentrating the polynucleic acids present in the sample; (ii) if need be amplifying the relevant part of the antibiotic resistance genes present in said sample with at least one suitable primer pair; (iii) hybridizing the polynucleic acids of step (i) or (ii) with at least one of the rpoB gene probes, as specified in table 2, under the appropriate hybridization and wash conditions; (iv) detecting the hybrids formed in step (iii); (v) inferring the Mycobacterium antibiotic resistance spectrum, and possibly the Mycobacterium species involved from the differential hybridization signal(s) obtained in step (iv).

[0001] The present invention relates to the field of drug-resistantmycobacteria.

[0002] The present invention relates to probes, primers, methods andkits comprising the same for the detection of mycobacterial nucleicacids in biological samples.

[0003] Identification of most clinically relevant Mycobacterium species,in particular of Mycobacterium tuberculosis is tedious and timeconsuming due to culture-procedures which can take up to 6 weeks. Rapiddiagnosis of Mycobacterium infection is very important since the diseasemight be life-threatening and highly contagious. Only recently somemethods—all making use of one or another amplification process—have beendeveloped to detect and identify Mycobacterium species without the needfor culture (Claridge et al. 1993). Most of these methods are still inevaluation and their benefit in routine applications remainsquestionable. Moreover, these methods do not solve the problem ofMycobacterium drug-resistance detection which still relies on culture.

[0004] Since the frequency of multidrug resistance in tuberculosis issteadily increasing (Culliton, 1992), it is clear now that earlydiagnosis of M. tuberculosis and the rapid recognition of resistance tothe major tuberculostatics are essential for therapy and an optimalcontrol of the resurgent epidemic.

[0005] The antibiotics used for treatment of M. tuberculosis infectionsare mainly isoniazid and rifampicini either administered separately oras a combination of both. Occasionally, pyrazinamide, ethambutol andstreptomycin are used: other classes of antibiotics like(fluoro)quinolones may become the preferred tuberculostatics in thefuture.

[0006] Since most multidrug resistant mycobacteria also lostsusceptibility to rifampicin, rifampicin-resistance is considered to bea potential marker for multidrug resistant tuberculosis. For thisreason, the detection of resistance to rifampicini might be ofparticular relevance.

[0007] For the majority of the M. tuberculosis strains examined so far,the mechanism responsible for resistance to rifampicin (and analogueslike rifabutin) has been elucidated. Rifampicin (and analogues) blockthe RNA polymerase by interacting with the β-subunit of this enzyme.Telenti et al. (1993a) found that mutations in a limited region of theβ-subunit of the RNA polymerase of M. tuberculosis give rise toinsensitivity of the RNA polymerase for rifampicin action. This regionis limited to a stretch of 23 codons in the rpoB gene. The authorsdescribe 17 amino acid changes provoking resistance to rifampicin(Telenti et al. 1993b). These amino acid changes are caused by pointmutations or deletions at 15 nucleotides or 8 amino acid codonsrespectively scattered over a stretch of 67 nucleotides or 23 amino acidcodons.

[0008] Telenti et al. (1993a and b) described a PCR-SSCP method toscreen for the relevant mutations responsible for rifampicin resistance(SSCP refers to single-strand conformation polymorphism). SSCP analysiscan be performed either by using radio-activity or by using fluorescentmarkers. In the latter case sophisticated and expensive equipment (anautomated DNA-sequencing apparatus) is needed. The SSCP approachdescribed has also other limitations with respect to specificity andsensitivity which might impede its routine use. Specimen can only beadequatly analysed directly if a significant load of bacteria(microscopy score: >90 organisms/field) is observed microscopically andon crude DNA samples strand-separation artefacts may be observed whichcomplicate the interpretation of the results.

[0009] Kapur et al. (1994) describe 23 distinct rpoB alleles associatedwith rifampicin resistance. In addition to the mutations described byTelenti et al. (1993a), some new mutant rpoB alleles are described.However, the most frequently occuring alleles remain the same as thosedescribed before.

[0010] In M. leprae, the molecular basis for rifampicin resistance wasdescribed by Honoré and Cole (1993). Here too, resistance stemmed frommutations in the rpoB gene, which encodes the beta subunit of RNApolymerase of M. leprae. Only a limited number of resistant M. lepraestrains (9) were analysed, and in most of them (8/9) resistance was dueto a mutation affecting the Ser-425 residue.

[0011] Clinically important mycobacteria other than M. tuberculosis andM. leprae often show an innate, be it variable, resistance torifampicin. This is the case for M. avium and M. intracellulare, humanpathogens for which only limited treatment options are available.Guerrero et al. (1994) compared the rpoB-gene sequences of different M.avium and M. intracellulare isolates with that of M. tuberculosis.Differences are present at the nucleotide level but a full amino acididentity was found with rifampicin-sensitive M. tuberculosis. Thesefindings suggest that another mechanism of resistance, possibly apermeability barrier, applies for M. avium and M. intracellulare.

[0012] The specific detection of point mutations or small deletions canelegantly be approached using hybridization procedures such as thereverse hybridization assay. However, the complexity observed in therelevant part of the rpoB gene does not allow a straightforward probedevelopment. As will be exemplified further, it was one of the objectsof the present invention to design a specific approach allowing thedetection of most if not all mutations found so far in a fast andconvenient way without the need for sophisticated equipment.

[0013] The mechanism of resistance to isoniazid (INH) is considerablymore complex than that for rifampicin. At least two gene products areinvolved in INH-resistance. First, there is catalase-peroxidase which isbelieved to convert INH to an activated molecule. Hence, strains whichdo not produce catalase-peroxidase by virtue of a detective or deletedkatG gene are not anymore susceptible to INH (Zhang et al. 1992:Stoeckle et al. 1993). In this context it should be mentioned that theassociation between INH-resistance and the loss of catalase activity wasalready noted in the fifties (Middlebrook, 1954 a and b; Youatt, 1969).

[0014] The second molecule involved is the inhA gene product, which isbelieved to play a role in the mycolic acid biosynthesis. It ispostulated that the activated INH molecule interacts either directly orindirectly with this product and probably prevents proper mycolic acidbiosynthesis. This hypothesis is based on the recent observation thatoverexpression of the wild type inhA gene or a particular amino acidchange (S94A) in the inhA gene product confers resistance to INH(Banerjee et al., 1994).

[0015] In short, and somewhat simplified we can state that in certain M.tuberculosis strains resistance to INH might be mediated by:

[0016] the loss of catalase-peroxidase activity

[0017] the presence of certain amino acid changes in the inhA protein

[0018] the expression level of the wild type inhA protein

[0019] Also, other mechanisms might be involved in confering resistanceto INH and related drugs. The importance of these factors in the totalspectrum of INH-resistance mechanisms has yet to be assessed. This issuecan be addressed by means of DNA probe techniques if reliable DNA probescan be developed from the available DNA-sequences of the katG gene (EMBLn° X68081) and inhA gene (EMBL n° U02492) of M. tuberculosis. Theseprobe-tests could then also be applied for detection of drug resistancein biological samples.

[0020] For the detection of resistance to streptomycine and(fluoro)quinolones the same approach as for rifampicin can be followed.Resistance to these antibiotics is also induced by point mutations in alimited region of one or more genes. Point mutations in the gyrase geneconfer resistance to (fluoro)quinolones (EMBL n° L27512). Streptomycinresistance is correlated with mutations in either the 16S rRNA gene orthe gene of a ribosomal protein S12 (rpsL) (Finken et al., 1993: Douglasand Steyn, 1993: Nair et al. 1993).

[0021] Resistance due to nucleotide changes in the katG, rpoB and rpsLgenes have been described in international application WO 93/22454. Foreach of the different genes in M. tuberculosis only one of the manypossible mutations was specified in detail, n1. R461L for katG. S425L(equivalent to S531L described by Telenti et al. and the presentinvention) for rpoB and K42R for rpsL.

[0022] It is an object of the present invention to provide a rapid andreliable detection approach for determination of antibiotic resistanceof mycobacterial species present in a biological sample.

[0023] More particularly, it is an aim of the present invention toprovide a rapid and reliable method for determination of resistance torifampicin (and/or rifabutin) of M. tuberculosis present in a biologicalsample.

[0024] It is also an object of the present invention to provide methodsenabling the detection and identification of Mycobacterium species in abiological sample, directly coupled to the monitoring of the antibioticresistance spectrum.

[0025] It is more particularely an aim of the present invention toprovide a method to detect the presence of Mycobacterium tuberculosis ina biological sample directly coupled to the detection of rifampicin,(and/or rifabutin) resistance.

[0026] It is more particularely an aim of the present invention toprovide a method to detect the presence of Mycobacterium leprae in abiological sample directly coupled to the detection of rifampicin(and/or rifabutin) resistance.

[0027] It is also an aim of the present invention to select particularprobes able to discriminate wild-type sequences from mutated sequencesconferring resistance to one or more drugs.

[0028] It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type sequences from mutatedsequences conferring resistance to rifampicin (and/or rifabutin).

[0029] It is more particularly an aim of the present invention to selecta particular set of probes, able to discriminate wild-type sequencesfrom mutated sequences conferring resistance to rifampicin (an/orrifabutin) with this particular set of probes being used under the samehybridisation and wash-conditions.

[0030] It is moreover an aim of the present invention to combine a setof selected probes able to discriminate wild-type sequences from mutatedsequences conferring resistance to rifampicin (and/or rifabutin) withanother set of selected probes able to identify the mycobacteria speciespresent in the biological sample, whereby all probes can be used underthe same hybridisation and wash-conditions.

[0031] It is also an aim of the present invention to select primersenabling the amplification of the gene fragment(s) determining theantibiotic resistance trait of interest.

[0032] It is more particularly an aim of the present invention to selectprimers enabling the amplification of the rpoB-gene fragment determiningresistance to rifampicin (and analogues).

[0033] Another aim of the invention is to provide kits for the detectionof antibiotic resistance in mycobacteria, possibly coupled to theidentification of the mycobacterial species involved.

[0034] All the aims of the present invention have been met by thefollowing specific embodiments.

[0035] The selection of the preferred probes of the present invention isbased on the Line Probe Assay (LiPA) principle which is a reversehybridization assay using oligonlucleotide probes immobilized asparallel lines on a solid support strip (Stuyver et al. 1993;international application WO 94/12670). This approach is particularlyadvantageous since it is fast and simple to perform. The reversehybridization format and more particularly the LiPA approach has manypractical advantages as compared to other DNA techniques orhybridization formats, especially when the use of a combination ofprobes is preferable or unavoidable to obtain the relevant informationsought.

[0036] It is to be understood, however, that any other type ofhybridization assay or format using any of the selected probes asdescribed further in the invention, is also covered by the presentinvention.

[0037] The reverse hybridization approach implies that the probes areimmobilized to a solid support and that the target DNA is labelled inorder to enable the detection of the hybrids formed.

[0038] The following definitions serve to illustrate the terms andexpressions used in the present invention.

[0039] The target material in these samples may either be DNA or RNAe.g. genomic DNA or messenger RNA or amplified versions thereof. Thesemolecules are also termed polynucleic acids.

[0040] The term “probe” refers to single stranded sequence-specificoligonucleotides which have a sequence which is complementary to thetarget sequence to be detected.

[0041] The term complementary as used herein means that the sequence ofthe single stranded probe is exactly complementary to the sequence ofthe single-stranded target, with the target being defined as thesequence where the mutation to be detected is located. Since the currentapplication requires the detection of single basepair mismatches, verystringent conditions for hybridization are required, allowing inprinciple only hybridization of exactly complementary sequences.However, variations are possible in the length of the probes (seebelow), and it should be noted that, since the central part of the probeis essential for its hybridization characteristics, possible deviationsof the probe sequence versus the target sequence may be allowabletowards head and tail of the probe, when longer probe sequences areused. These variations, which may be conceived from the common knowledgein the art, should however always be evaluated experimentally, in orderto check if they result in equivalent hybridization characteristics thanthe exactly complementary probes.

[0042] Preferably, the probes are about 5 to 50 nucleotides long, morepreferably from about 10 to 25 nucleotides. The nucleotides as used inthe present invention may be ribonucleotides, deoxyribonucleotides andmodified nucleotides such as inosine or nucleotides containing modifiedgroups which do not essentially alter their hybridisationcharacteristics. Probe sequences are represented throughout thespecification as single stranded DNA oligonucleotides from the 5′ to the3 end. It is obvious to the man skilled in the art that any of thebelow-specified probes can be used as such, or in their complementaryform, or in their RNA form (wherein T is replaced by U).

[0043] The probes according to the invention can be prepared by cloningof recombinant plasmids containing inserts including the correspondingnucleotide sequences, if need be by cleaving the latter out from thecloned plasmids upon using the adequate nucleases and recovering them,e.g. by fractionation according to molecular weight. The probesaccording to the present invention can also be synthesized chemically,for instance by the conventional phospho-triester method.

[0044] The term “solid support” can refer to any substrate to which anoligonucleotide probe can be coupled, provided that it retains itshybridization characteristics and provided that the background level ofhybridization remains low. Usually the solid substrate will be amicrotiter plate, a membrane (e.g. nylon or nitrocellulose) or amicrosphere (bead). Prior to application to the membrane or fixation itmay be convenient to modify the nucleic acid probe in order tofacilitate fixation or improve the hybridization efficiency. Suchmodifications may encompass homopolymer tailing, coupling with differentreactive groups such as aliphatic groups, NH₂ groups, SH groups,carboxylic groups, or coupling with biotin, haptens or proteins.

[0045] The term “labelled” refers to the use of labelled nucleic acids.Labelling may be carried out by the use of labelled nucleotidesincorporated during the polymerase step of the amplification such asillustrated by Saiki et al. (1988) or Bei et al. (1990) or labelledprimers, or by any other method known to the person skilled in the art.The nature of the label may be isotopic (³²P, ³⁵S, etc.) or non-isotopic(biotin, digoxigenin, etc.).

[0046] The term “primer” refers to a single stranded oligonucleotidesequence capable of acting as a point of initiation for synthesis of aprimer extension product which is complementary to the nucleic acidstrand to be copied. The length and the sequence of the primer must besuch that they allow to prime the synthesis of the extension products.Preferably the primer is about 5-50 nucleotides long. Specific lengthand sequence will depend on the complexity of the required DNA or RNAtargets, as well as on the conditions of primer use such as temperatureand ionic strenght.

[0047] The fact that amplification primers do not have to match exactlywith the corresponding template sequence to warrant proper amplificationis amply documented in the literature (Kwok et al., 1990).

[0048] The amplification method used can be either polymerase chainreaction (PCR; Saiki et al. 1988), ligase chain reaction (LCR: Landgrenet al., 1988; Wu & Wallace, 1989; Barany, 1991), nucleic acidsequence-based amplification (NASBA; Guatelli et al., 1990; Compton,1991), transcription-based amplification system (TAS; Kwoh et al.,1989), strand displacement amplification (SDA; Duck. 1990; Walker et al.1992) or amplification by means of Qβ replicase (Lizardi et al. 1988;Lomeli et al., 1989) or any other suitable method to amplify nucleicacid molecules known in the art.

[0049] The oligonucleotides used as primers or probes may also comprisenucleotide analogues such as phosphorothiates (Matsukura et al., 1987),alkylphosphorothiates (Miller et al. 1979) or peptide nucleic acids(Nielsen et al. 1991; Nielsen et al., 1993), or may containintercalating agents (Asseline et al. 1984).

[0050] As most other variations or modifications introduced into theoriginal DNA sequences of the invention these variations willnecessitate adaptions with respect to the conditions under which theoligonucleotide should be used to obtain the required specificity andsensitivity. However the eventual results of hybridisation will beessentially the same as those obtained with the unmodifiedoligonucleotides.

[0051] The introduction of these modifications may be advantageous inorder to positively influence characteristics such as hybridizationkinetics, reversibility of the hybrid-formation, biological stability ofthe oligonucleotide molecules, etc.

[0052] The “sample” may be any biological material taken either directlyfrom the infected human being (or animal), or after culturing(enrichment). Biological material may be e.g. expectorations of anykind, broncheolavages, blood, skin tissue, biopsies, lymphocyte bloodculture material, colonies, liquid cultures, soil, faecal samples, urineetc.

[0053] The probes of the invention are designed for attaining optimalperformance under the same hybridization conditions so that they can beused in sets for simultaneous hybridization; this highly increases theusefulness of these probes and results in a significant gain in time andlabour. Evidently, when other hybridization conditions would bepreferred, all probes should be adapted accordingly by adding ordeleting a number oft nucleotides at their extremities. It should beunderstood that these concommitant adaptations should give rise toessentially the same result, namely that the respective probes stillhybridize specifically with the defined target. Such adaptations mightalso be necessary if the amplified material should be RNA in nature andnot DNA as in the case for the NASBA system.

[0054] For designing probes with desired characteristics, the followinguseful guidelines known to the person skilled in the art can be applied.

[0055] Because the extent and specificity of hybridization reactionssuch as those described herein are affected by a number of factors,manipulation of one or more of those factors will determine the exactsensitivity and specificity of a particular probe, whether perfectlycomplementary to its target or not. The importance and effect of variousassay conditions, explained further herein, are known to those skilledin the art.

[0056] First, the stability of the [probe: target] nucleic acid hybridshould be chosen to be compatible with the assay conditions. This may beaccomplished by avoiding long AT-rich sequences, by terminating thehybrids with G:C base pairs, and by designing the probe with anappropriate Tm. The beginning and end points of the probe should bechosen so that the length and %GC result in a Tm about 2-10° C. higherthan the temperature at which the final assay will be performed. Thebase composition of the probe is significant because G-C base pairsexhibit greater thermal stability as compared to A-T base pairs due toadditional hydrogen bonding. Thus, hybridization involving complementarynucleic acids of higher G-C content will be stable at highertemperatures.

[0057] Conditions such as ionic strenght and incubation temperatureunder which a probe will be used should also be taken into account whendesigning a probe. It is known that hybridization will increase as theionic strenght of the reaction mixture increases, and that the thermalstability of the hybrids will increase with increasing ionic strenght.On the other hand, chemical reagents, such as formamide, urea, DMSO andalcohols, which disrupt hydrogen bonds, will increase the stringency ofhybridization. Destabilization of the hydrogen bonds by such reagentscan greatly reduce the Tm. In general, optimal hybridization forsynthetic oligonucleotide probes of about 10-50 bases in length occursapproximately 5° C. below the melting temperature for a given duplex.Incubation at temperatures below the optimum may allow mismatched basesequences to hybridize and can therefore result in reduced specificity.

[0058] It is desirable to have probes which hybridize only underconditions of high stringency. Under high stringency conditions onlyhighly complementary nucleic acid hybrids will form; hybrids without asufficient degree of complementarity will not form. Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity needed between two nucleic acid strands forming ahybrid. The degree of stringency is chosen such as to maximize thedifference in stability between the hybrid formed with the target andthe nontarget nucleic acid. In the present case, single base pairchanges need to be detected, which requires conditions of very highstringency.

[0059] Second, probes should be positioned so as to minimize thestability of the [probe: nontarget] nucleic acid hybrid. This may beaccomplished by minimizing the length of perfect complementarity tonon-target organisms, by avoiding GC-rich regions of homology tonontarget sequences, and by positioning the probe to span as manydestabilizing mismatches as possible. Whether a probe sequence is usefulto detect only a specific type of organism depends largely on thethermal stability difference between [probe:target] hybrids and[probe:nontarget] hybrids. In designing probes, the differences in theseTm values should be as large as possible (e.g. at least 2° C. andpreferably 5° C.).

[0060] The length of the target nucleic acid sequence and, accordingly,the length of the probe sequence can also be important. In some cases,there may be several sequences from a particular region, varying inlocation and length, which will yield probes with the desiredhybridization characteristics. In other cases, one sequence may besignificantly better than another which differs merely by a single base.While it is possible for nucleic acids that are not perfectlycomplementary to hybridize, the longest stretch of perfectlycomplementary base sequence will normally primarily determine hybridstability. While oligonucleotide probes of different lengths and basecomposition may be used, preferred oligonucleotide probes of thisinvention are between about D to 50 (more particularely 10-25) bases inlength and have a sufficient stretch in the sequence which is perfectlycomplementary to the target nucleic acid sequence.

[0061] Third, regions in the target DNA or RNA which are known to formstrong internal structures inhibitory to hybridization are lesspreferred. Likewise, probes with extensive self-complementarity shouldbe avoided. As explained above, hybridization is the association of twosingle strands of complementary nucleic acids to form a hydrogen bondeddouble strand. It is implicit that if one of the two strands is whollyor partially involved in a hybrid that it will be less able toparticipate in formation of a new hybrid. There can be intramolecularand intermolecular hybrids formed within the molecules of one type ofprobe if there is sufficient self complementarity. Such structures canbe avoided through careful probe design. By designing a probe so that asubstantial portion of the sequence of interest is single stranded, therate and extent of hybridization may be greatly increased. Computerprograms are available to search for this type of interaction. However,in certain instances, it may not be possible to avoid this type ofinteraction.

[0062] The present invention provides in its most general form for amethod to detect the antibiotic resistance spectrum of Mycobacteriumspecies present in a sample, possibly coupled to the identification ofthe Mycobacterium species involved, comprising the steps of:

[0063] (i) if need be releasing, isolating or concentrating thepolynucleic acids present in the sample;

[0064] (ii) if need be amplifying the relevant part of the antibioticresistance genes present in said sample with at least one suitableprimer pair;

[0065] (iii) hybridizing the polynucleic acids of step (i) or (ii) withat least one of the rpoB gene probes as mentioned in table 2, underappropiate hybridization and wash conditions;

[0066] (iv) detecting the hybrids formed in step (iii);

[0067] (v) inferring the Mycobacterium antibiotic resistance spectrum,and possibly the Mycobacterium species involved from the differentialhybridization signal(s) obtained in step (iv).

[0068] The relevant part of the antibiotic resistance genes refers tothe regions in the p B, katG, inhA, 16S rRNA, rpsL and gyrase genesharboring mutations causing resistance to rifampicin, isoniazid,streptomycin and (fluoro)quinolones as described above.

[0069] According to a preferred embodiment of the present invention,step (iii) is performed using a set of probes meticulously designed assuch that they show the desired hybridization results, when used underthe same hybridization and wash conditions.

[0070] More specifically, the present invention provides a method fordetection of rifampicin (and/or rifabutin) resistance of M. tuberculosispresent in a biological sample, comprising the steps of:

[0071] (i) if need be releasing, isolating or concentrating thepolynucleic acids present in the sample;

[0072] (ii) if need be amplifying the relevant part of the rpoB genepresent in said polynucleic acids with at least one suitable primerpair:

[0073] (iii) hybridizing the polynucleic acids of step (i) or (ii) witha selected set of rpoB wild-type probes under appropiate hybridizationand wash conditions, with said set comprising at least one of thefollowing probes (see Table 2): S1 (SEQ ID NO 1) S11 (SEQ ID NO 2) S2(SEQ ID NO 3) 53 (SEQ ID NO 4) S33 (SEQ ID NO 5) S4 (SEQ ID NO 6) S44(SEQ ID NO 7) S444 (SEQ ID NO 43) S4444 (SEQ ID NO 8) S5 (SEQ ID NO 9)S55 (SEQ ID NO 10) S555 (SEQ ID NO 39) S5555 (SEQ ID NO 40) S55C (SEQ IDNO 44) S55M (SEQ ID NO 45) S6 (SEQ ID NO 11) S66 (SEQ ID NO 12)

[0074] (iv) detecting the hybrids formed in step (iii);

[0075] (v) inferring the rifampicin susceptibility (sensitivity versusresistance) of M. tuberculosis present in the sample from thedifferential hybridization signal(s) obtained in step (iv).

[0076] The term susceptibility refers to the phenotypic characteristicof the M. tuberculosis strain being either resistant or sensitive to thedrug, as determined by in vitro culture methods, more specifically torifampicin (and/or rifabutin). Resistance to rifampicin is revealed bythe absence of hybridization with at least one of the S-probes.

[0077] Standard hybridization and wash conditions are for instance 3×SSC(Sodium Saline Citrate). 20% deionized FA (Formamide) at 50° C. Othersolutions (SSPE (Sodium saline phosphate EDTA), TMACI (Tetramethylammonium Chloride), etc.) and temperatures can also be used providedthat the specificity and sensitivity of the probes is maintained. Ifneed be, slight modifications of the probes in length or in sequencehave to be carried out to maintain the specificity and sensitivityrequired under the given circumstances. Using the probes of theinvention, changing the conditions to 1.4×SSC. 0.07% SDS at 62° C. leadto the same hybridisation results as those obtained under standardconditions, without the necessity to adapt the sequence or length of theprobes.

[0078] Suitable primer pairs can be chosen from a list of primer pairsas described below.

[0079] In a more preferential embodiment, the above-mentionedpolynucleic acids from step (i) or (ii) are hybridized with at leasttwo, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen or seventeen of the above-mentionedS-probes, preferably with 5 or 6 S-probes, which, taken together, coverthe “mutation region” of the rpoB gene.

[0080] The term “mutation region” means the region in the rpoB-genesequence where most, if not all of the mutations responsible forrifampicin resistance are located. This mutation region is representedin FIG. 1.

[0081] In a more preferential embodiment, the above-mentionedpolynucleic acids from step (i) or (ii) are hybridized with a selectedset of rpoB-wild-type probes, with said set comprising at least one, andpreferentially all of the following probes (see Table 2): S11 (SEQ ID NO2) S2 (SEQ ID NO 3) S33 (SEQ ID NO 5) S4444 (SEQ ID NO 8) S55 or S5555(SEQ ID NO 10 or 40)

[0082] In another particular embodiment the set of S-probes as describedabove, or at least one of them, can be combined with one or moreSIL-probes, detecting silent mutations in the rpoB gene. A preferentialSIL-probe is SIL-1 (SEQ ID NO 13, see Table 2).

[0083] In another embodiment of the invention, the set of S-probes andpossibly SIL-probes, can be combined with at least one R-probe detectinga specific mutation associated with rifampicin-resistance.

[0084] R-probes are selected from the following group of probes (seeTable 2): R1 (SEQ ID NO 46) R2 (SEQ ID NO 14) R2B (SEQ ID NO 47) R2C(SEQ ID NO 48) R3 (SEQ ID NO 49) R4A (SEQ ID NO 15) R44A (SEQ ID NO 16)R444A (SEQ ID NO 17) R4B (SEQ ID NO 18) R44B (SEQ ID NO 19) R444B (SEQID NO 20) R4C (SEQ ID NO 50) R4D (SEQ ID NO 51) R4E (SEQ ID NO 52) R5(SEQ ID NO 21) R55 (SEQ ID NO 22) R5B (SEQ ID NO 53) R5C (SEQ ID NO 54)

[0085] Preferably the set of S-probes and possibly SIL-probes can becombined with at least two, three, four, five, six, or more R probes.

[0086] Most preferably, the set of S-probes and possibly SIL-probes arecombined with at least one R-probe from the following restricted groupof probes R2 (SEQ ID NO 14) R444A (SEQ ID NO 17) R444B (SEQ ID NO 20)R55 (SEQ ID NO 22)

[0087] In the case where S and R probes are combined, resistance torifampicin is revealed by absence of hybridization with one of theS-probes and possibly by a positive hybridization signal with thecorresponding R-probe.

[0088] Since some mutations may be more frequently occurring thanothers, e.g. in certain geographic areas (see e.g. Table 5) or inspecific circumstances (e.g. rather closed communities) it may beappropiate to screen only for specific mutations, using a selected setof S and/or R probes. This would result in a more simple test, whichwould cover the needs under certain circumstances. According to Telentiet al. (1993a and b) most mutations described in his publication arerelatively rare (3% or less): predominant mutations are S531L (51.6%).H526Y (12.5%), D516V (9.4%) and H1526D (7.8%).

[0089] In a particular embodiment of the invention a selected set of twoor three S-probes is used, the respective sets of probes being: S4444(SEQ ID NO 8) S55 (or S5555) (SEQ ID NO 10 or 40) S2 (SEQ ID NO 3) S4444(SEQ ID NO 8) S55 (or S5555) (SEQ ID NO 10 or 40)

[0090] or

[0091] Using this restricted sets of S-probes the majority of rifampicinresistant cases can be detected, by an absence oft hybridisation signalwith at least one of these probes.

[0092] In another particular embodiment of the invention, at least one Rprobe is used, possibly combined with a selected set of two or three Sprobes as described above, said R probe being chosen from the followinglist of probes: R2 (SEQ ID NO 14) R444A (SEQ ID NO 17) R444B (SEQ ID NO20) R55 (SEQ ID NO 22)

[0093] In this case, the specific mutation responsible for therifampicin-resistant phenotype can be inferred from a positivehybridization signal with one of the R-probes and/or the absence ofhybridization with the corresponding S-probe.

[0094] In another embodiment of the invention, the above-mentioned S,SIL or R probes may be combined with at least one species-specific probefor M. tuberculosis allowing simultaneous identification ofMycobacterium tuberculosis and detection of rifampicin resistance, withsaid species-specific probe being chosen from the following group ofprobes (see Table 2): MT-POL-1 (SEQ ID NO 23) MT-POL-2 (SEQ ID NO 24)MT-POL-3 (SEQ ID NO 25) MT-POL-4 (SEQ ID NO 26) MT-POL-5 (SEQ ID NO 27)

[0095] Most preferably the species-specific M. tuberculosis probe is:

[0096] MT-POL-1 (SEQ ID NO 23)

[0097] In vet another embodiment of the invention, the above-mentionedS, SIL, R or, MT-POL probes can be combined with at least onespecies-specific probe for M. paratuberculosis, M. avium, M.scrophulaceum, M. kansasii, M. intracellulare (and MAC-strains) or M.leprae, with said probes being respectively MP-POL-1 (SEQ ID NO 28),MA-POL-1 (SEQ ID NO 29), MS-POL-1 (SEQ ID NO 38), MK-POL-1 (SEQ ID NO55), MI-POL-1 (SEQ ID NO 68) and ML-POL-1 (SEQ ID NO 57) (see table 2B)or any species-specific probe derived from the sequence of the relevantpart of the rpoB gene of M. paratuberculosis (SEQ ID NO 35), M. avium(SEQ ID NO 36), M. scrofulaceum (SEQ ID NO 37), M. kansasii (SEQ ID NO56) or MAC-strains (SEQ ID NO 69), as represented respectively in FIGS.5, 6, 7, 8 and 11. It should be noted that the sequences represented inFIGS. 5-8 and 11 are new. The sequence of the rpoB-gene fragment of M.intracellulare and M. leprae has been described by others (Guerrero etal. 1994; Honoré and Cole, 1993).

[0098] The term “MAC-strains” means “M. avium complex” strains known tothe man skilled in the art of mycobacteria taxonomy. This ratherheterogeneous group of MAC-strains may however comprise strains whichare genotypically rather like M. intracellulare. This is also the casefor isolate ITG 926, of which the rpoB sequence is shown in FIG. 11. TheMI-POL-1 probe derived from SEQ ID NO 69 and the published M.intracellulare rpoB sequence is therefor specific for M. intracellulareand some MAC strains together.

[0099] It should be stressed that all of the above-mentioned probes,including the species-specific probes, are contained in the sequence ofthe rpoB gene, and more particulary in the sequence of the amplifiedrpoB gene fragment. Moreover, as illustrated further in the examples,the probes described above as “preferential”, are designed in such a waythat they can all be used simultaneously, under the same hybridizationand wash conditions. These two criteria imply that a singleamplification and hybridization step is sufficient for the simultaneousdetection of rifampicin resistance and the identification of themycobacterial species involved.

[0100] In a preferential embodiment, and by wart of an example, a methodis disclosed to detect M. tuberculosis and its resistance to rifampicin,comprising the steps of:

[0101] (i) if need be releasing, isolating or concentrating thepolynucleic acids present in the sample;

[0102] (ii) if need be amplifying the relevant part of the rpoB genewith at least one suitable primer pair;

[0103] (iii) hybridizing the polynucleic acids of step (i) or (ii) withthe following set of probes under appropriate hybridization and washconditions

[0104] MT-POL-1

[0105] S11

[0106] S33

[0107] S4444

[0108] S55 or S5555

[0109] R2

[0110] R444A

[0111] R444B

[0112] R55

[0113] (iv) detecting the hybrids formed in step (iii);

[0114] (v) inferring the presence of M. tuberculosis and itssusceptibility to rifampicin from the differential hybridizationsignal(s) obtained in step (iv).

[0115] In order to detect the mycobacterial organisms and/or theirresistance pattern with the selected set of oligonucleotide probes, anyhybridization method known in the art can be used (conventionaldot-blot, Southern blot, sandwich, etc.).

[0116] However, in order to obtain fast and easy results if a multitudeof probes are involved, a reverse hybridization format may be mostconvenient.

[0117] In a preferred embodiment the selected set of probes areimmobilized to a solid support. In another preferred embodiment theselected set of probes are immobilized to a membrane strip in a linefashion. Said probes may be immobilized individually or as mixtures todelineated locations on the solid support.

[0118] A specific and very user-friendly embodiment of theabove-mentioned preferential method is the LiPA method, where theabove-mentioned set of probes is immobilized in parallel lines on amembrane, as further described in the examples.

[0119] The above mentioned R-probes detect mutations which have alreadybeen described in the prior art (Telenti et al., 1993a and 1993b).However, as illustrated further in the examples, four new mutationsassociated with rifampicin-resistance in M. tuberculosis, not yetdescribed by others, are disclosed by the current invention. Using theS-probes of the current invention, new mutations as well as mutationsalready described in the prior art can be detected. The unique conceptof using a set of S-probes covering the complete mutation region in therpoB gene, allows to detect most, if not all of the mutations in therpoB-gene responsible for rifampicin resistance, even those mutationswhich would not yet have been described uptil now.

[0120] The four new rpoB mutations (D516G, H526C, H526T and R529Q) aremarked in Table 1 with an asterisk. By way of an example, the sequenceof the SB-mutant allele H526C is represented in FIG. 2 (SEQ ID NO 34).

[0121] The invention also provides for any probes or primersets designedto specifically detect or amplify specifically these new rpoB genemutations, and any method or kits using said primersets or probes.

[0122] In another embodiment, the invention also provides for a methodfor detection of rifampicin (and/or rifabutin) resistance of M. lepraepresent in a biological sample, comprising the steps of:

[0123] (i) if need be releasing, isolating or concentrating thepolynucleic acids present in the sample;

[0124] (ii) if need be amplifying the relevant part of the rpoB genewith at least one suitable primer pair;

[0125] (iii) hybridizing the polynucleic acids of step (i) or (ii) witha selected set of rpoB wild-type probes under appropiate hybridizationand wash conditions, with said set comprising at least one of thefollowing probes (see Table 2): ML-S1 (SEQ ID NO 58) ML-S2 (SEQ ID NO59) ML-S3 (SEQ ID NO 60) ML-S4 (SEQ ID NO 61) ML-S5 (SEQ ID NO 62) ML-S6(SEQ ID NO 63)

[0126] (iv) detecting the hybrids formed in step (iii);

[0127] (v) inferring the rifampicin susceptibility (sensitivity versusresistance) of M. leprae present in the sample from the differentialhybridization signal(s) obtained in step (iv).

[0128] Resistance to rifampicin is revealed by the absence ofhybridization with at least one of the ML-S-probes.

[0129] In another embodiment of the invention, the above-mentioned ML-Sprobes may be combined with a species specific probe for M. leprae.ML-POL-1, allowing simultaneous identification of M. leprae anddetection of rifampicin resistance, with said species specific probebeing represented in SEQ ID NO 57.

[0130] It is to be noted that the above-mentioned ML-S probes andML-POL-1 probe are all contained within the same amplified rpoB-genefragment of M. leprae, and are designed as such that they can all beused under the same hybridization and wash conditions.

[0131] The invention further provides for any of the probes as describedabove, as wemm as compositions comprising at least one of these probes.

[0132] The invention also provides for a set of primers, allowingamplification of the mutation region of the rpoB gene of M.tuberculosis. The sets of primers can be chosen from the following groupof sets (see table 2): P1 and P5 (SEQ ID NO 30 and 33) P3 and P4 (SEQ IDNO 31 and 32) P7 and P8 (SEQ ID NO 41 and 42) P2 and P6, in combinationwith (P1 and P5) or (P3 and P4) or (P7 and P8) Most preferably, the setof primers is the following: P3 and P4 (SEQ ID NO 31 and 32).

[0133] The invention also provides for a set of primers allowingamplification of the mutation region of the rpoB gene in mycobacteria ingeneral, i.e. in at least M. tuberculosis, M. avium, M.paratuberculosis, M. intracellulare, M. leprae, M. scrofulaceuin. Thesegeneral primers can be used e.g. in samples where the presence oftmycobacteria other than M. tuberculosis is suspected, and where it isdesirable to have a more general detection method.

[0134] The set of primers is composed of a 5′-primer, selected from thefollowing set: MGRPO-1 (SEQ ID NO 64) MGRPO-2 (SEQ ID NO 65)

[0135] and a 3′-primer, selected from the following set: MGRPO-3 (SEQ IDNO 66) MGRPO-4 (SEQ ID NO 67).

[0136] The sequence of these primers is shown in Table 2B.

[0137] Primers may be labeled with a label of choice (e.g. biotine).Different primer-based target amplification systems may be used, andpreferably PCR-amplification, as set out in the examples. Single-roundor nested PCR may be used.

[0138] The invention also provides for a kit for inferring theantibiotic resistance spectrum of mycobacteria present in a biologicalsample, possibly coupled to the identification of the mycobacterialspecies involved, comprising the following components:

[0139] (i) when appropiate, a means for releasing, isolating orconcentrating the polynucleic acids present in the sample;

[0140] (ii) when appropriate, at least one of the above-defined set ofprimers;

[0141] (iii) at least one of the probes as defined above, possibly fixedto a solid support;

[0142] (iv) a hybridization buffer, or components necessary forproducing said buffer;

[0143] (v) a wash solution, or components necessary for producing saidsolution;

[0144] (vi) when appropriate, a means for detecting the hybridsresulting from the preceding hybridization.

[0145] The term “hybridization buffer” means a buffer enabling ahybridization reaction to occur between the probes and the polynucleicacids present in the sample, or the amplified products, under theappropiate stringency conditions.

[0146] The term “wash solution” means a solution enabling washing of thehybrids formed under the appropiate stringency conditions.

[0147] More specifically, the invention provides for a kit as describedabove, for the simultaneous detection of M. tuberculosis and itsresistance to rifampicin.

[0148] In another specific case, the invention provides for a kit asdescribed above, for the simultaneous detection of M. leprae and itsresistance to rifampicin.

[0149] Table Legends

[0150] Table 1 summarizes the nucleotide and amino acid chances(described by Telenti et al. (1993a and b), Kapur et al. 1994 and thepresent invention) in the rpoB gene fragment of rifampicin resistant M.tuberculosis isolates. Codon numbering is as in FIG. 1. New mutationsdescribed by the current invention are indicated with an asterisk (*).

[0151] Table 2 lists the sequences of the primers and probes selectedfrom the rpoB gene.

[0152] 2A: M. tuberculosis

[0153] 2B: other mycobacterial species

[0154] Table 3 shows the hybridization results obtained with probeMT-POL-1, with DNA from different mycobacterial and non-mycobacterialspecies.

[0155] Table 4 shows some representative results obtained with LiPA forsome M. tuberculosis isolates which have been sequenced and theinterpretation of the different LiPA patterns.

[0156] Table 5 shows the occurrence of the different rpoB mutations inM. tuberculosis in different geographical areas. Abbreviations:Bel=Belium, Bengla=Bengladesh, Bur-Fa=Burkina faso, Buru=Burundi,Can=Canada, Chi=Chili, Col=Colombia, Egy=Egypt, Gui=Guinea,Hon=Honduras, Pak=Pakistan, Rwa=Rwanda, Tun=Tunesia.

[0157] Table 6 shows a comparison of LiPA results versus rifampicinresistance determination in culture for M. tuberculosis. S=Sensitive,R=Resistant.

FIGURE LEGENDS

[0158]FIG. 1 represents the nucleotide sequence and the amino acidsequence of the mutation region of respectively the rpoB gene and theRNA polymerase B-subunit of a wild-type (i.e. not resistant)Mycobacterium tuberculosis strain (ITG 9081). The codon (amino acid)numbering is as in Telenti et al. (1993a). The nucleotides or aminoacids involved in resistance-inducing changes are underlined. Theobserved mutations are boxed. The horizontal bars indicate the positionsof some of the oligonucleotide probes (one probe per group isindicated).

[0159]FIG. 2 shows the partial nucleotide sequence of the newlydescribed rpoB mutant allele of M. tuberculosis strain ITG 9003 (SEQ IDNO 34).

[0160]FIG. 3 shows results obtained on LiPA-strips with probes S44 andS4444 applied at different concentrations on the membrane strip. Astarget material, nucleic acid preparations of M. tuberculosis strainsITG 8872 and ITG 9081 were used.

[0161]FIG. 4 gives a comparison of the performance of probes S44 andS4444 in a Line probe assay conformation. The probes on strip A and Bare identical except for S44 and S4444. The hybridizations wereperformed using amplified material originating from M. tuberculosisstrain ITG 8872.

[0162]FIG. 5 shows the partial nucleotide sequence of the presumptiverpoB gene of M. paratuberculosis strain 316F (SEQ ID NO 35)

[0163]FIG. 6 shows the partial nucleotide sequence of the presumptiverpoB gene of M. aviuim strain ITG 5887 (SEQ ID NO 36)

[0164]FIG. 7 shows the partial nucleotide sequence of the presumptiverpoB gene of M. scrofulaceum strain ITG 4979 (SEQ ID NO 37)

[0165]FIG. 8 shows the partial nucleotide sequence of the presumptiverpoB gene of M. kansasii strain ITG 4987 (SEQ ID NO 56)

[0166]FIG. 9 shows some rpoB mutations in M. tuberculosis and theircorresponding LiPA patterns. Nomenclature of the mutations is asdescribed in Table 1. Nomenclature of the LiPA pattern is as follows:

[0167] wt=positive hybridization with all S-probes, and negativehybridization with all R-probes;

[0168] ΔS1-5=absence of hybridization with the respective S-probe;

[0169] R2, 4A, 4B, 5=positive hybridization with the respective R-probesand absence of hybridization with the corresponding S-probe;

[0170] C=positive control line: should be positive when the test iscarried out properly;

[0171] Mtb=MT-POL-probe.

[0172] Only one probe of each group is mentioned, but it stands for thewhole group: e.g. S5 stands for S5. S55, S555, S5555, S55C, S55M.

[0173]FIG. 10 shows the frequency of the different mutations encounteredin the rifampicin-resistant M. tuberculosis strains analysed by LiPA.Nomenclature is as in FIG. 9. “Mix” means that a mix of strains waspresent in the sample. “Double” means the presence of two mutations inone strain.

[0174]FIG. 11 shows the partial nucleotide sequence of the presumptiverpoB gene of MAC strain ITG 926 (SEQ ID NO 69).

EXAMPLE 1 Amplification of the rpoB Gene Fragment in M. tuberculosis

[0175] After nucleic acid extraction from mycobacterial isolates (eithercultured or present in body fluid or tissue) 2 μl product was used toamplify the relevant part of the rpo B gene by using one or morecombinations between the 5′-primers (P1 (SEQ ID NO 30), P2, P3 (SEQ IDNO 31) and P7 (SEQ ID NO 41)) and the 3′-primers (P4 (SEQ ID NO 32). P5(SEQ ID NO 33), P6 and P8 (SEQ ID NO 42).

[0176] The sequence of these primers is given in Table 2, P1, P3, P4,P5, P7 and P8 are new primer sequences, described by the currentinvention. P2 and P6 have been described before (Telenti et al. 1993a).

[0177] These primers may be labeled with a label of choice (e.g.biotine). Different primer-based target-amplification systems may beused. For amplification using the PCR, the conditions used are describedhereafter. Single-round amplification with primers P1 and P5 involved 35cycles of 45 sec/94° C., 45 sec/58° C. 45 sec/72° C. If a nested PCR waspreferable, in the second round primers P3 and P4 were used and 25cycles (45 sec/94° C. 60 sec/68° C.) were performed starting from 1 μlof first round product.

[0178] If P3 and P4 were used in a single-round PCR, the followingcycling protocol was used: 30 cycles of 1 min/95° C., 1 min/55° C., 1min/72° C. The same cycling protocol was used for set of primers P2/P6.

[0179] PCRs were usually performed in a total volume of 50 HI containing50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.2 mM MgCl₂, 200 μl each dNTP,0.01% gelatine and 1 U Taq polymerase.

[0180] Primer concentrations ranged between 10 and 25 pmol/reaction.

[0181] Since the set of primers P3/P4 yielded significantly strongersignals after hybridization than the set of primers P2/P6, the first setof primers was used for all further hybridization experiments. Set ofprimers P2/P6 was used for sequence analysis. Nested PCR using P2 and P6as outer primers and P3 and P4 as inner (biotinylated) primers was onlyused for direct detection in clinical samples (expectorations andbiopsies).

[0182] The length of the amplified product, as monitored byagarose-gelelectrophoresis, is as follows:

[0183] Primer set P1/P5: 379 basepairs

[0184] Primer set P3/P4: 257 basepairs

[0185] Primer set P2/P6: 411 basepairs

[0186] Primer set P7/P8: 339 basepairs

EXAMPLE 2 Sequencing of rpoB Gene Fragments from M. tuberculosis Strains

[0187] DNA extracted from mycobacterial isolates known to be resistantor sensitive to rifampicin was amplified using the set of primers P1/P6(of which P6 was biotinylated at the 5′ end). Direct sequencing ofsingle stranded PCR-product was performed by using streptavidin coatedbeads and the Taq Dye Deoxy Terminator I Cycle Sequencing kit on anAB1373A DNA sequencer (Applied Biosystems, Forster City, Calif. USA) asrecommended by the manufacturer. The primers used for sequencing werethe same than those used for amplification (P2 or P6).

[0188] As expected, all sensitive (=sensitive in culture and sensitiveLiPA pattern) strains sequenced (7) yielded a wild-type sequence (nomutation). In most resistant strains a mutation could be identified.Most of these mutations have been previously described (Telenti et al.1993a, 1993b, Kapur et al. 1994). However, 4 new mutations are describedin the current invention: D516G, H526C. H526T and R529Q (see also Table1 (*)). The full sequence of the amplified rpoB fragment of mutantallele H526C (isolate ITG 9003) is shown in FIG. 2 (SEQ ID NO 34) by wayof an example.

[0189] A few strains (3/180, see table 6) were resistant to rifampicinin culture but showed a wild-type LiPA pattern. After sequencing, theseisolates all showed a wild-type rpoB gene sequence, confirming thehybridization results. It is therefore possible that for these isolatesa different molecular mechanism of rifampicin resistance applies.

EXAMPLE 3 Development of the Line Probe Assay (LiPA)-Strip

[0190] The principle and protocol of the line probe assay was asdescribed earlier (Stuyver et al. 1993) with a few exceptions. Insteadof the incorporation of biotinylated dUTP, biotinylated primers wereused and the hybridization and stringent wash were performed at 50° C.in 3×SSC/20% deionized formamide.

[0191] The mutations in the rpoB gene leading to resistance torifampicin are mainly located in a small area of the gene spanning 67nucleotides (23 amino acid codons). At least 17 nucleotides, evenlyintersperced over this region, are involved in mutations leading to atleast 28 different amino acid changes. Such a complexity of nucleotidechanges poses problems for the detection of all these changes in asingle hybridization step. In principle, per mutated site one wild-typeprobe would be needed and per nucleotide change one mutant-probespecifically detecting that particular mutation. Hence, a hybridizationtest would involve at least about 35 sequence specific oligonucleotideprobes which would render this test complex to manufacture and use, andconsequently commercially less attractive.

[0192] Therefore a particular approach was designed making use ofcarefully chosen wild-type (S-) probes, each spanning more than onepolymorphic site and overlapping the complete area of relevance (seeFIG. 1). Doing so, the set of at least 10 wild-type probes could bereduced to a total of five or six probes. These probes were meticulouslyadjusted experimentally such that the presence of each resistancecausing mutation in the rpoB mutation region resulted in a clearlydetectable decrease in hybrid-stability between the target and at leastone of these probes under the same hybridization and wash conditions.

[0193] Since with this combination of probes all relevant mutationsdescribed so far can be detected, rifampicin resistance can be monitoredin Mycobacterium tuberculosis isolates. This set of probes is also ableto detect the presence of non-described mutations, e.g. the TGC mutationat position 526 in strain ITG 9003.

[0194] Although strictly taken the addition of mutant (R-) probes to theselected panel of wild-type probes is obsolete, for scientific purpossesit might be informative to detect the exact point mutation present.Therefore, 4 mutant probes (R2, R4A, R4B, R5) were designed whichcorrespond to the most frequently occurring mutations and which, takentogether, are able to positively identify more than 70% of all resistantcases (see FIG. 10). Additional mutant probes (e.g. R1, R2B, R2C, R3,R4C, R4D, R4E, R5B, R5C) may be added to this set of 4 in order topositively identify most clinically relevant resistant cases. Theaddition of these probes also adds to the reliability of the assay sincethe appearance of a mutant probe should inevitably result in thedisappearance of a wild-type probe if one is not dealing with mixedinfections. In some cases, it may be of clinical relevance todistinguish between the different mutations possibly present. Thissituation may occur for instance at amino-acid position 516. If at thatposition the normal (wild-type) amino acid present (Aspartic acid),changes into a Valine or Tyrosine, high level resistance or intermediatelevel resistance to rifampicin is induced; however, unpublished dataseem to indicate that the sensitivity to rifabutin is maintained. Hence,for infections by strains with this mutation, rifabutin would still beeffective where as rifampicin is not. The same may be true for codon 511but, to our knowledge, these are the only mutation-sites for which theeffect of rifampicin and rifabutin might be different; usually strainsresistant to rifampicin are also resistant to rifabutin.

[0195] In order to develop a LiPA-strip to detect the presence ofmutations generating resistance to rifampicin (and/or rifabutin) in M.tuberculosis a total of 36 oligonucleotide probes were synthesized andevaluated in a reverse hybridization test. The sequence of these probesis shown in Table 2A (wild-type and mutation probes). The first set ofprobes tested was: S1, S2, S3, S4, S5, R2, R4A, R4B and R5. Under theconditions used for reverse hybridization most probes did not perform astheoretically expected and modifications had to be introduced leading tothe synthesis and evaluation of the following additional probes: S11,S33, S44, S4444, S5555, R44A, R444A, R44B, R444B, R55. From the totalpanel of probes, probes exhibiting the most optimal features withrespect to specificity and sensitivity, under the same experimentalconditions were selected for further use.

[0196] The prefered probes with wild-type sequences (S-probes) whichtogether overlap the entire rpoB region of interest are: S11, S2, S33,S4444 and S55 or S5555 (see Table 2). Resistance will be detected by aloss of hybridization signal with any of these S-probes.

[0197] The prefered mutation probes (R-probes) are: R2, R444A, R444B andR55 (see table 2). In some cases of resistance a loss of hybridizationsignal with the S-probes may be accompanied by a positive hybridizationsignal with the corresponding R-probe.

[0198] By way of an example, some of the rpoB-mutations and theircorresponding LiPA pattern are shown in FIG. 9.

[0199] Although the probes from the same group (e.g. probes S4, S44,S444 and S4444) are differing only slightly from each other, theirhybridization characteristics may vary considerably. This is illustratedby way of example in an experiment in which the performance of probesS44 and S4444 is compared using PCR products originating from two M.tuberculosis strains (ITG 8872 and ITG 9081) both displaying a wild-typesequence in the target region of probes S44 and S4444. The differencebetween both probes is shown below S44 GGTTGACCCACAAGCGCCGA  |||||||||||||||||| S4444   TTGACCCACAAGCGCCGACTGTC

[0200] Probe S44 was chosen on a theoretical basis from the known genesequence (Telenti et al. 1993a). This probe would be theoretically thebest probe for discriminating mismatches in the CAC-codon (underlined)since this codon is in the central part of the probe. However, incontrast to the general rules, the performance of probe S4444 proved tobe significantly better (as described in the following experiment).

[0201] Both probes were applied to nitrocellulose strips in differentamounts (2.4, 1.2 and 0.6 pmollstrip). After fixation and blocking ofthe strips, the probes were hybridized with biotinylated PCR fragments(from strains IGT 8872 and ITG 9081) as described earlier. The resultsare shown in FIG. 3.

[0202] Under the hybridization conditions used (3×SSC. 20% deionizedformamide. 50° C.) the signals obtained with probe S44 are very weak. Onthe other hand probe S4444 generates very strong and reliable signals.Consequently, probe S4444 is prefered over S44 for use on theLiPA-strip. This dramatic effect cannot only be attributed to thedifference in length of both probes, but also the location seems to beof importance. Most probably the secundary structure of the targetregion of the probes highly influences the hybridizationcharacteristics.

[0203] In FIG. 4 the results of another experiment are shown in whichthe performance of both probes is compared in the context of the otherprobes. Both strips A and B are identically processed using amplifiedproduct originating from strain ITG 8872. Both strips are almostidentical except that strip A contains probe S44 (0.6 pmol) and strip Bprobe S4444 (0.1 pmol) and the order of the probes applied to the stripis different. On strip A probe S44 is negative whereas on strip B probeS4444 is clearly positive, although in both cases there is a perfectmatch between the target and the probe and in both cases a positiveresult would have been expected.

[0204] These results clearly illustrate that probe design, especiallyunder the fixed conditions of the reverse hybridization format (the sameconditions for each probe) is not straightforward and probes have to beevaluated meticulously before they can be used in a reversehybridization format.

[0205] In general we can state that suitable probes cannot always besimply derived on a theoretical basis from a known gene sequence.

[0206] Although not detected among the isolates tested in the currentinvention, the presence of a silent mutation may give rise to aresistant pattern on the strips (one wild-type probe missing) while thestrain is sensitive. Up to now only one silent substitution has beendescribed (in codon 528: CGC→CGT: Telenti et al. 1993a). This mutationwould result in a destabilization of the hybrid with probe S-4444.However, by adding to the strip a probe specific for hie silent mutation(Probe SIL-1, Table 2), this silent mutation can be discriminated fromresistance inducing mutations and would prevent misinterpretation of thepattern observed. Moreover, the SIL-probes can be applied on the stripat the same location than the corresponding S-probes (mixed probes).Doing so, no loss of hybridisation signal would be observed as a resultof the silent mutation.

[0207] In order to detect the insertion mutations conferring rifampicinresistance, 514insF and 514insFM (see FIG. 1), two new wild-type probeswere designed. S6 and S66, the sequence of which is represented in Table2. Hybridization with nucleic acids originating from strains harbouringthese insertion mutations, results in the absence of a hybridizationsignal with S6 and S66 (see also Table 1).

[0208] In order to positively identify more mutations than thosedetected by the set of R2, R4A, R4B and R5, a number of additionalR-probes were designed, which may be added to the LiPA strip, using thesame hybridization and wash conditions. Together with theabove-described R-probes, the additional R-probes (R1, R2B, R2C, R3,R4C, R4D, R4E, R5B, R5C) allow a positive identification of themutations most frequently encountered in the strain collection tested inthe current application.

EXAMPLE 4 Probe Specific for the M. tuberculosis Complex

[0209] A line probe assay was developed in order to enable thesimultaneous detection of the mutations causing resistance to rifampicindirectly coupled to detection of the pathogen in casu M. tuberculosis.

[0210] Since it is extremely advantageous to be able to detectsimultaneously the presence of M. tuberculosis and the presence orabsence of a gene or mutation causing drug-resistance, the aim was todevelop a M. tuberculosis probe contained in the same PCR fragment as atleast one of the relevant resistance markers to be identified. ThereforerpoB genes of non-M. tuberculosis isolates were sequenced. Theseorganisms were: M. paratuberculosis 316F. M. avium ITG5887, M.scrofulaceum ITG4979 and M. kansasii ITG4987. The sequences of thecorresponding rpoB gene fragments are shown in FIGS. 5 to 8,respectively. In addition, the sequence of the rpoB gene fragments of M.leprae and M. intracellulare were known from the literature (Hlonoré andCole. 1993; Guerrero et al. 1994). Comparison of these sequences withthe rpoB gene sequence of M. tuberculosis enabled the delineation of aspecific region (outside the region responsible for resistance) in therpoB gene fragment from which the development of probes, potentiallyspecific for M. tuberculosis appeared feasible. An oligonucleotide probe(further referred to as MT-POL-1) derived from that region with thefollowing sequence:

[0211] GGA CGT GGA GGC GAT CAC ACC (SEQ ID NO 23)

[0212] was further evaluated with respect to its sensitivity andspecificity by hybridization on LiPA strips. The amplification andhybridization conditions were as described above. The results aresummarized in Table 3. In addition to the four M. tuberculosis strainslisted in Table 3, 521 more M. tuberculosis clinical isolates weretested: all isolates gave a positive hybridization signal. Evidently,also M. bovis strains hybridize with the probe, but it is clinically notrelevant to distinguish M. tuberculosis from M. bovis for this purpose.In fact, throughout the application the term “M. tuberculosis” can bereplaced by “M. tuberculosis complex”, referring to M. tuberculosiss.s., M. bovis, M. africanum and M. microti, without affecting thesignificance of the results.

[0213] Although, using the sets of primers described in example 1, a PCRproduct may be obtained with DNA of some other Mycobacterium species andeven some genetically unrelated micro-organisms which might also bepresent in the respiratory tract, none of these bacteria showedhybridization with the selected MT-POL-1 probe. In conclusion we canstate that the selected probe is highly specific for M. tuberculosiscomplex strains and 100% sensitive for M. tuberculosis. Also otherprobes from this region might be useful for the specific detection of M.tuberculosis complex strains, such as: MT-POL-1, MT-POL-3, MT-POL-4 andMT-POL-5 (see Table 2).

[0214] In a similar way, the following probes can be used todifferentiate M. avium, M. paratuberculosis, M. scrofulaceum, M.kansasii, M. intracellulare and M. leprae strains from each other andfrom other mycobacteria MA-POL-1, MP-POL-1, MS-POL-1, MK-POL-1, MI-POL-Iand ML-POL-I respectively (see table 2B).

EXAMPLE 5 Evaluation of LiPA Strips for M. tuberculosis

[0215] LiPA strips were prepared carrying the following probes (inaddition to a positive control line): MT-POL-1, S11, S2, S33, S4444,S5555, R2, R444A, R444B, R55.

[0216] These strips were hybridized with PCR products from M.tuberculosis strains for which the relevant rpoB gene sequence wasdetermined.

[0217] Some representative results are summarized in Table 4.

[0218] The hybridization results completely correlated with thesequencing results, indicating that the probes used can discriminate atthe level of a single mismatch. Each mutation screened for could bedetected either by the absence of one of the wild-type probes (S-probes)or by the absence of a wild-type probe together with the presence of thecorresponding mutant probe (R-probe). In the latter case, the exactmutation present can be derived from the hybridization results. However,the knowledge of the exact mutation present is not necessary todetermine whether or not one is dealing with a strain resistant torifampicin since each mutation screened for confers resistance.Sensitive strains, i.e. strains without mutations in the relevant partof the rpoB gene, give positive reactions on all S-probes while allR-probes score negative (=wt-pattern).

EXAMPLE 6 Direct Detection of M. tuberculosis Strains andRifampicin-Resistance in Clinical Samples

[0219] Sixty-eight clinical specimens from different geographicalorigins (13 and 35 sputum specimens from Belgium and Rwandarespectively, and 20 lymph node biopsies from Burundi) all positive inculture for M. tuberculosis and kept at −20° C. were analyzed. Samplepreparation for amplification was based on the procedure of Boom et al.(1990) modified by De Beenhouwer et al. (submitted). A nested PCRapproach for the relevant region of the rpoB gene was carried out withbiotinylated inner primers (P3 and P4). After thermal cycling, theamplified product was incubated with the LiPA strip. Rifampicinresistance was determined on Löwenstein-Jensen using the proportionmethod of Canetti et al. (1963). For resistant strains, the MIC (MinimalInhibitory Concentration) of rifampicin was determined on 7H10 agar(Heifets, 1988).

[0220] Microscopically, 20 (29.4%) samples were negative, 15 (22.1%)weakly positive (1+ or less according to the scale of the AmericanThoracic Society) and 33 (48.5%) strongly positive (≧+2) uponZiehl-Neelsen staining.

[0221] The LiPA detected rifampicin sensitivity in 49 specimens (only M.tuberculosis and wild-type probes positive) and resistance in 19specimens (M. tuberculosis probe positive, one of the wild-type probesmissing, and eventually one of the mutation probes positive). In vitrorifampicin susceptibility testing confirmed these results with threeexceptions. For all the sensitive strains, a sensitive probe pattern wasobserved indicating that possible silent mutations were not detected inthis series. All strains displaying a resistance pattern were found tobe resistant by conventional techniques. For three specimens (from threemultidrug resistant patients from Rwanda) a sensitive pattern wasobtained by PCR-LiPA while culture revealed resistance (MIC>2 μg/ml on7H10). Sequencing of the rpoB region of these strains confirmed thewild-type gene sequence, possibly implying a different mechanism ofrifampicin resistance in these cases or a mutation in another part ofthe rpoB-gene. It is also interesting to note that the nested PCR systemgave positive results for all the tested specimens positive in cultureincluding 20 Ziehl-Neelsen negative specimens. In another experiment(data not shown) including 17 smear negative sputum specimens fromclinically suspected tuberculosis cases negative in culture, no signalat all was obtained with the LiPA system, indicating that the infectionswere most probably not caused by M. tuberculosis.

[0222] In a subsequent experiment, a large collection of clinicalspecimens from different geographical origin were tested in LiPA.Results are shown in Table 5. Of the 137 resistant strains found, quitea large number could be attributed to one of the mutations representedby the R-probes (R2, R4A, R4B, R5). Interestingly, some mutations seemto be more frequent in some countries as compared to others (e.g. inTunesia and Egypt; R4B (=H526D), in Rwanda; R5 (S531L)). This masteventually lead to different test formats for different countries.

[0223] On a total of 213 rifampicin-resistant strains analysed by LiPAin the current application, 151 (71%) could be attributed to themutations S531L, H526D, D516V or H526Y, and were thus detectable by apositive signal with respectively probes R5, R4B, R2 and R4A (see FIG.10).

[0224] On a total of 180 strains analysed both by culture and by LiPA,correct identification (sensitive/resistant) was made in 164 (=91.1%) ofthe strains (see Table 6). In three resistant strains. LiPA results andsequencing showed a wild-type rpoB-gene fragment, which indicates thatthe mechanism for rifampicin resistance could not be attributed tomutations in the investigated part of the rpoB gene.

[0225] Thirteen of the 180 analyzed strains were resistant in LiPA butseemed to be sensitive in culture. However, after reculturing 2 of these13 strains in synthetic 7H11 medium in stead of the traditionalLöwenstein Jensen medium, they turned out to be rifampicin resistantanyway. This uptil now unpublished finding shows that the conventionalLöwenstein Jensen medium is not recommended for determination ofantibiotic susceptibility of mycobacteria (possibly due to the presenceof traces of antibiotics found in commercially available eggs used toprepare Löwenstein Jensen medium). Therefore, the percentage ofdiscrepant strains which are resistant in LiPA but sensitive in culture(in this case 13/180=7.2%) is expected to be much lower (and possibly0%) when culturing is done on a synthetic medium like 7H11.

[0226] Interestingly, most of the rifampicin resistant isolates (>90%)examined in the current application were in addition resistant toisoniazid, and thus multidrug-resistant (definition of multidrugresistance=resistant to at least isoniazid and rifampicin). Rifampicinresistance can thus be considered as a potential marker for multidrugresistance, and the above-described LiPA test may therefore be animportant tool for the control of multidrug resistant tuberculosis.

[0227] In conclusion, the above-described method permits correctidentification of M. tuberculosis and simultaneous detection ofrifampicin resistance directly in clinical specimens withoutpreculturing. It enables easy detection of rifampicin resistancedirectly in a clinical specimen in less than 24 hours.

EXAMPLE 7 Detection of Rifampicin Resistance in M. leprae

[0228] The above-described detection approach can also be applied to thedetection of M. leprae present in biological samples coupled to thedetection of its resistance to rifampicin. The sequence of the rpoB geneof M. leprae was described earlied by Honoré and Cole (1993). They onlyidentified a limited number of mutations responsible for rifampicinresistance in M. leprae. It can be reasonably expected that, similar toM. tuberculosis, a lot of other mutations may cause rifampicinresistance in M. leprae, and that most of these mutations will belocalized in a rather restricted area of the rpoB gene, corresponding tothe “mutation region” described earlier in this application.

[0229] Therefore, a set of wild-type probes is selected overlapping theputative mutation region in the rpoB-gene of M. leprae (see table 2B):ML-S1 (SEQ ID NO 58) ML-S2 (SEQ ID NO 59) ML-S3 (SEQ ID NO 60) ML-S4(SEQ ID NO 61) ML-S5 (SEQ ID NO 62) ML-S6 (SEQ ID NO 63).

[0230] Rifampicin resistance is revealed by an absence of hybridizationwith at least one of these ML-S probes. This set of ML-S probes willdetect rifampicin-resistance causing mutations in this region, eventhough the sequence of these mutations has not yet been specified.

[0231] The above-mentioned ML-S probes have been carefully designed insuch a way that they can all be used under the same hybridization andwash conditions. The same holds for the species specific ML-POL-1 probe,with which the ML-S probes can be combined to allow a simultaneousdetection of M. leprae and its resistance to rifampicin.

[0232] All the probes mentioned in this example are contained in thesame rpoB gene fragment of M. leprae, which can be obtained by PCR usinga set of primers chosen from MGRPO-1 or MGRPO-2 (5′ primers) and MGRPO-3or MGRPO-4 (3′ primers). TABLE 1 Probe detection Position Wild-typesequence Mutation sequence Δ Wt Mutant Abbr. Nucl. Codon Nucl. Codon AANucl. Codon AA probe probe L511P  9 511 T CTG Leu C CCG Pro S11 — L511R 9 511 T CTG Leu G CGG Arg S11 — S512T 12 512 G AGC Ser C ACC Thr S11 —Q5131 15 513 A CAA Gln T CTA Leu S11 — Q513K 14 513 C CAA Gln A AAA LysS11 — 514 ins F 16 514 — — — TTC TTC Phe S6 — 514 ins FM 16 514 — — —TTC ATG TTC ATG Phe S6 — Met Δ514-516 15-23 514-516 AA TTC ATG G AA TTCATG G Gln Δ Δ His S1-S2- — Phe S6 Met Asp Δ516-517 23-28 516-517 GAC CAGGAC CAG Asp Δ Δ Δ S2-S6 — Gln D516Y 23 516 G GAC Asp T TAC Tyr S2 —D516V 24 516 A GAC Asp T GTC Val S2 R2 D516E 25 516 C GAC Asp G GAG GluS2 — D516G(*) 24 516 A GAC Asp G GGC Gly S2 — Δ517-518 26-31 517-518 CAGAAC CAG AAC Gln Δ Δ Δ S2 — Asn Δ518 29-31 518 AAC AAC Asn Δ Δ Δ S2 —S522L 42 522 C TCG Ser T TTG Leu S33 — H526Y 53 526 C CAC His T TAC TyrS4444 R444A H526D 53 526 C CAC His G GAC Asp S4444 R444B H526N 53 526 CCAC His A AAC Asn S4444 — H526C(*) 53-54 526 CA CAC His TG TGC Cys S4444— H526R 54 526 A CAC His G CGC Arg S4444 — H526P 54 526 A CAC His C CCCPro S4444 — H526Q 55 526 C CAC His ^(A) _(G) CA^(A) _(G) Gln S4444 —H526L 54 526 A CAC His C CTC Leu S4444 — H526T(*) 53-54 526 CA CAC HisAC ACC Thr S4444 — R529Q(*) 63 529 G CGC Arg A CAA Gln S4444 — S531Q68-69 531 TC TCG Ser CA CAG Gln S5555 — S531L 69 531 C TCG Ser T TTG LeuS5555 R55 S531W 69 531 C TCG Ser G TGG Trp S5555 — S531Y 69-70 531 CGTCG Ser A^(T) _(C) TA^(T) _(C) Tyr S5555 — S531C 69-70 531 CG TCG Ser GTTGT Cys S5555 — L533P 75 533 T CTG Leu C CCG Pro S5555 —

[0233] TABLE 2 Primers and probes selected from the rpoB gene SEQ ID NO2A: M. tuberculosis Primers P1 GAGAATTCGGTCGGCGAGCTGATCC 30 P2TACGGTCGGCGAGCTGATCC P3 GGTCGGCATGTCGCGGATGG 31 P4 GCACGTCGCGGACCTCCAGC32 P5 CGAAGCTTGACCCGCGCTACACC 33 P6 TACGGCGTTTCGATGAACC P7CGGCATGTCGCGGATGGAGCG 41 P8 CGGCTCGCTGTCGGTGTACGC 42 Probesspecies-specific probes MT-POL-1 GGACGTGGAGGCGATCACACC 23 MT-POL-2CGATCACACCGCAGACGTTGATC 24 MT-POL-3 CATCCGGCCGGTGGTCGC 25 MT-POL-4CTGGGGCCCGGCGGTCT 26 MT-POL-5 CGGTCTGTCACGTGAGCGTG 27 wild-type probesS1 CAGCCAGCTGAGCCAATTCATG  1 S11 CAGCCAGCTGAGCCAATTCAT  2 S2TTCATGGACCAGAACAACCCGC  3 S3 AACCCGCTGTCGGGGTTGA  4 S33AACCCGCTGTCGGGGTTGACC  5 S4 GTTGACCCACAAGCGCCGA  6 S44GGTTGACCCACAAGCGCCGA  7 S444 TTGACCCACAAGCGCCGACTGT 43 S4444TTGACCCACAAGCGCCGACTGTC  8 S55 CGACTGTCGGCGCTGGGGC 10 S555GACTGTCGGCGCTGGGGCC 39 S5555 GACTGTCGGCGCTGGGGC 40 S55CGCCCCAGCGCCGACAGTCG 44 S55M CGACTGTCGGCGTTGGGGC 45 S6TGAGCCAATTCATGGACCAGAA 11 S66 CTGAGGCAATTCATGGACCAGA 12 SIL-1CCACAAGCGTCGACTGTCG 13 mutant probes R2 AATTCATGGTCCAGAACAACCCG. 14 R4AGGTTGACCTACAAGCGCCGA 15 R44A GGGTTGACCTACAAGCGCCGA 16 R444ATTGACCTACAAGCGCCGACTGTC 17 R4B GTTGACCGACAAGCGCCGA 18 R44BGGTTGACCGACAAGCGCCGA 19 R444B TTGACCGACAAGCGCCGACTGTC 20 R5CGACTGTTGGCGCTGGGG 21 R55 CGACTGTTGGCGCTGGGGC 22 R1CAGCCAGCCGAGCCAATTCAT 46 R2B AATTCATGTACCAGAACAACCCG 47 R2CATGGACCAGAACCCGCTGTCG 48 R3 AACCCGCTGTTGGGGTTGACC 49 R4CTTGACCCGCAAGCGCCGACTGTC 50 R4D TTGACCCCCAAGCGCCGACTGTC 51 R4ETTGACCTGCAAGCGCCGACTGTG 52 R5B CGACTGTGGGCGCTGGGGC 53 R5CACTGTGGGCGGCGGGGCCC 54 2B: other mycobacterial species primers MGRPO-1CCAAAACCAGATCCGGGTCGG 64 MGRPO-2 GTCCGGGAGCGGATGACCAC 65 MGRPO-3GGGTGCACGTCGCGGACCTC 66 MGRPO-4 GGGCACATCCGGCCGTAGTG 67 probes MP-POL-1CATCCGTCCCGTCGTGGC 28 MA-POL-1 CATCCGTCCAGTCGTGGCG 29 MS-POL-1GCCGGTCGTGGCCGCG 38 MK-POL-1 AGCGCCGGCTTTCGGCGC 55 ML-POL-1GACGCTGATCAATATCCGTCCGG 57 MI-POL-1 ATCCGGCCGGTCGTCGCC 68 ML-S1:CAGCCAGCTGTCGCAGTTCATG 58 ML-S2: GTTCATGGATCAGAACAACCCTC 59 ML-S3:AACCCTCTGTGGGGCCTGACC 60 ML-S4: ACCCAGAAGCGCCGGCTGTC 61 ML-S5:GGCTGTCGGCGCTGGGC 62 ML-S6: CTGTCGCAGTTCATGGATCAGA 63

[0234] TABLE 3 Hybridization results obtained with probe MT-POL-1Species Strain MT-POL-1 1 M. tuberculosis ATCC 27294 + 2 M. tuberculosisNCTC 7417 + 3 M. tuberculosis ITG 8017 + 4 M. tuberculosis ITG 9173 + 5M. bovis BCG (Kopen) + 6 M. bovis patient isolate + 7 M. avium ITG 5872− 8 M. avium ITG 5874 − 9 M. intracellulare ITG 5913 − 10 M.intracellulare ITG 5918 − 11 M. paratuberculosis 2 E − 12 M. kansasiiITG 4987 − 13 M. marinum ITG 7732 − 14 M. scrofulaceum ITG 4988 − 15 M.gordonae ITG 4989 − 16 Bordetella pertussis NCTC 8189 − 17 Bordetellaparapertussis NCTC 7385 − 18 Bordetella bronchiseptica NCTC 8761 − 19Moraxella catarrhalis LMG 5128 − 20 Moraxella catarrhalis LMG 1133 − 21Moraxella catarrhalis LMG 4200 − 22 Moraxella catarrhalis LMG 4822 − 23Haemophilus inhluenzae NCTC 8143 − 24 Haemophilus intluenzae ITG 3877 −25 Streptococcus pneumoniae H90-11921 − 26 Streptococcus pneumoniaeH91-04493 − 27 Streptococcus pneumoniae H90-11780 − 28 Pseudomonascepacia ATCC 25609 − 29 Acinetobacter ATCC 23055 − calcoaceticus 30Staphylococcus aureus 6420 − 31 Staphylococcus aureus 6360 − 32Pseudomonas aeruginosa 5682 − 33 Pseudomonas aeruginosa 5732 −

[0235] TABLE 4 Interpretation of LiPA-results for rifampicin resistancedetection in M. tuberculosis Mutation MT- LiPA (verified by POL-1 S11 S2S33 S4444 S55 R2 R444A R444B R55 pattern sequencing)Interpretation + + + + + + − − − − wt − Sensitive + − + + + + − − − −ΔS1 in S1 Resistant + + − + + + − − − − ΔS2 in S2 Resistant + +− + + + + − − − R2 D516V Resistant + + + − + + − − − − ΔS3 in S3Resistant + + + + − + − − − − ΔS4 in S4 Resistant + + + + − + − + − −R4A II526Y Resistant + + + + − + − − + − R4B II526D Resistant + + + + +− − − − − ΔS5 in S5 Resistant + + + + + − − − − + R5 S531L Resistant

[0236] TABLE 5 Occurrence of different mutations in M. tuberculosisstrains originating from different countries # Mutation Bel Bengla BeninBur-Fa Buru Can Chi Col Egy Gui Hon Pak Rwa Tun 2 ΔS1 1 1 2 ΔS2 1 1 4ΔS3 1 3 19 ΔS4 1 1 2 2 1 1 1 8 2 8 ΔS5 1 1 2 1 2 1 11 R2 2 1 2 6 15 R4a4 1 1 2 2 1 2 2 14 R4b 1 1 3 1 8 56 R5 9 1 1 1 1 1 6 26 10 1 R2 + ? 1 2R4a + R5 1 1 1 R4b + R5 1 1 ΔS1/R2 1 1 ΔS1/ΔS2 1 137 Totaal 17 6 1 6 0 36 3 6 3 0 12 41 33

[0237] TABLE 6 Comparison of LiPA results versus rifampicin resistancedetermination in culture for M. tuberculosis. Culture S R LiPA S 71  3 R13 93

REFERENCES

[0238] Asseline J. Delarue M. Lancelot G. Toulme F. Thuong N (1984)Nucleic acid-binding molecules with high affinity and base sequencespecificity: intercalating agents covalently linked tooligodeoxynucleotides Proc. Natl. Acad. Sci. USA 81(11):3297-301.

[0239] Banerjee A., Dubnau E. Quernard A. et al. inhA, a gene encoding atarget for Isoniazid and Ethionamide in Mycobacterium tuberculosis.Science 1994: 263: 227-30.

[0240] Barany F. Genetic disease detection and DNA amplification usingcloned thermostable ligase. Proc Natl Acad Sci USA 1991: 88: 189-193.

[0241] Bej A, Mahbubani M. Miller R. Di Cesare J. Haff L. Atlas R.Mutiplex PCR amplification and immobilized capture probes for detectionof bacterial pathogens and indicators in water. Mol Cell Probes 1990:4:353-365.

[0242] Boom R., Sol C. J. A., Salimans M. M. M. et al. Rapid and simplemethod for purification of nucleic acids. J Clin Microbiol 1990: 28:495-503.

[0243] Canetti G. Froman S., Grosset J. et al. Mycobacteria: laboratorymethods for testing drug sensivity and resistance. Bull WHO 1963: 29:565-79.

[0244] Claridge J. E., Sliawar R. M., Shinnick T. M. and Plikaytis B. B.Large-scale use of polymerase schain reaction for detection ofMycobacterium tuberculosis in a routine mycobacteriology laboratory. JClin Microbiol 1993; 31: 2049-56.

[0245] Compton J. Nucleic acid sequence-based amplification. Nature1991: 350: 91-92.

[0246] Culliton B. Drug resistant Th may bring epidemic. Nature 1992:356: 473.

[0247] Douglas J. Steyn L. M. A ribosomal gene mutation instreptomycin-resistant Mycobacterium tuberculosis isolates. J. Infect.Dis. 1993: 167: 1505-1506.

[0248] Duck P. Probe amplifier system based on chimeric cyclingoligonucleotides. Biotechniques 1990; 9: 142-147.

[0249] Finken M., Kirschner P., Meier A. Wrede A. and Böttger E.Molecular basis of streptomycin resistance in Mycobacteriumtuberculosis: Alternations of the ribosomal protein S12 gene and pointmutations within a functional 16S ribosomal RNA pseudoknot. MolMicrobiol 1993: 9: 1239-46.

[0250] Guatelli J, Whitfield K. Kwoh D. Barringer K. Richman D. GengerasT. Isothermal, in vitro amplification of nucleic acids by a multienzymereaction modeled after retroviral replication. Proc Natl Acad Sci USA1990: 87: 1874-1878.

[0251] Guerrero et al. Evaluation of the rpoB gene in rifampicinsusceptible and resistant M. avium and M. intracellulare. J. Antimicrob.Chemotherapy 1994: 33: 661-663.

[0252] Heifets L. Qualitative and quantitative drug-susceptibility testsin mycobacteriology. Pulmonary perspective. Am Rev Respir Dis 1988: 137:1217-1227.

[0253] Honoré N. and Cole S. Molecular basis of rifampicin resistance inM. leprae. Antimicrobial Agents and Chemotherapy 1993: 37: 414-418.

[0254] Kapur V. Li L. Iordanescu S. et al. Characterization by automatedDNA sequencing of mutations in the genie (rpoB) encoding the RNApolymerase β subunit in rifampicin resistant Mycobacterium tuberculosisstrains from New York City and Texas. J. Clin. Microbiol. 1994: 32:1095-1098.

[0255] Kwoh D. Davis G. Whitfield K. Chappelle H. Dimichele L. GingerasT. Transcription-based amplification system and detection of amplifiedhuman immunodeficiency virus type I with a bead-based sandwichhybridization format. Proc Natl Acad Sci USA 1989: 86: 1173-1177.

[0256] Kwok S. Kellogg D. McKinney N. Spasic D. Goda L. Levenson C.Sinisky J. Effects of primer-template mismatches on the polymerase chainreaction: Human immunodeficiency views type I model studies. Nucl. AcidsRes. 1990; 18: 999.

[0257] Landgren U. Kaiser R. Sanders J. Hood L. A ligase-mediated genedetection technique. Science 1988: 241:1077-1080.

[0258] Lomeli H. Tyagi S. Printchard C. Lisardi P. Kramer F.Quantitative assays based on the use of replicatable hybridizationprobes. Clin Chem 1989: 35: 1826-1831.

[0259] Matsukura M. Shinozuka K, Zon G. Mitsuya H. Reitz M. Cohen J.Broder S (1987) Phospliorothioate analogs of oligodeoxynucleotides:inhibitors of replication and cytopathic effects of humanimmunodeficiency virus. Proc. Natl. Acad. Sci. USA 84(21):7706-10.

[0260] Middlebrook G. (1954a). Isoniazid-resistance and catalaseactivity of tubercle bacilli. Am. Rev. Tuberc., 69: 471-472.

[0261] Middlebrook G., Cohn M. L., Schaefer W. B. (1954b). Studies onisoniazid and tubercle bacilli. III. The isolation, drug-susceptibility,and catalase-testing of tubercle bacilli from isoniazid-treatedpatients. Am. Rev. Tuberc., 70: 852-872.

[0262] Miller P, Yano J. Yano E. Carroll C. Jayaram K. Ts'o P (1979)Nonionic nucleic acid analogues. Synthesis and characterization ofdideoxyribonucleoside methylphosphonates. Biochemistry 18(23):5134-43.

[0263] Nair J. Rouse D A, Bai G H, Morris S L. The rpsL gene andstreptomycine resistance in single and multiple drug-resistant strainsor Mycobacterium tuberculosis. Mol. Microbiol. 1993: 10: 521-527.

[0264] Nielsen P. Egholm M, Berg R, Buchardt O (1991) Sequence-selectiverecognition of DNA by strand displacement with a thymine-substitutedpolyamide. Science 254(5037): 1497-500.

[0265] Nielsen P, Egholm M. Berg R. Buchardt O (1993) Sequence specificinhibition of DNA restriction enzyme cleavage by PNA. Nucleic-Acids-Res.21(2):197-200.

[0266] Saiki R, Walsh P, Levenson C. Erlich H. Genetic analysis ofamplified DNA with immobilized sequence-specific oligonucleotide probesProc Natl Acad Sci USA 1989: 86:6230-6234.

[0267] Stoeckle M Y, Guan L. Riegler N et al. Catalase-peroxidase genesequences in Isoniazid-sensitive and -resistant strains of Mycobacteriumtuberculosis from New York City. J. Inf. Dis. 1993; 168: 1063-65.

[0268] Stuyver L. Rossau R. Wyseur A. et al. Typing of hepatitis C virusisolates and characterization of new subtypes using a line probe assay.J. Gen. Virol. 1993; 74: 1093-1102.

[0269] Telenti A., Imboden P., Marchesi F. et al. Detection ofrifampicin-resistance mutations in Mycobacterium tuberculosis Lancet1993a; 341: 647-50.

[0270] Telenti A., Imboden P. Marchesi F. et al. Direct, automateddetection of Rifampin-resistant Mycobacterium tuberculosis by polymerasechain reaction and simple-strand conformation polymophism analysis.Antimicrob Agents Chemother 1993b: 37: 2054-58.

[0271] Wu D, Wallace B. The ligation amplification reaction(LAR)—amplification of specific DNA sequences using sequential rounds oftemplate-dependent ligation. Genomics 1989: 4:560-569.

[0272] Youatt J. (1969). A review of the action of isoniazid. Am. Rev.Respir. Dis. 99 729-749.

[0273] Zhang Y., Heym B. Allen B., Young D. and Cole S. Thecatalase-peroxidase gene and isoniazid resistance of Mycobacteriumtuberculosis. Nature 1992: 358: 591-93.

1 73 1 22 DNA Artificial Sequence Probe 1 cagccagctg agccaattca tg 22 221 DNA Artificial Sequence Probe 2 cagccagctg agccaattca t 21 3 22 DNAMycobacterium sp. 3 ttcatggacc agaacaaccc gc 22 4 19 DNA ArtificialSequence Probe 4 aacccgctgt cggggttga 19 5 21 DNA Artificial SequenceProbe 5 aacccgctgt cggggttgac c 21 6 19 DNA Artificial Sequence Probe 6gttgacccac aagcgccga 19 7 20 DNA Artificial Sequence Probe 7 ggttgacccacaagcgccga 20 8 23 DNA Artificial Sequence Probe 8 ttgacccaca agcgccgactgtc 23 9 17 DNA Artificial Sequence Probe 9 gactgtcggc gctgggg 17 10 19DNA Artificial Sequence Probe 10 cgactgtcgg cgctggggc 19 11 22 DNAArtificial Sequence Probe 11 tgagccaatt catggaccag aa 22 12 22 DNAArtificial Sequence Probe 12 ctgagccaat tcatggacca ga 22 13 19 DNAArtificial Sequence Probe 13 ccacaagcgt cgactgtcg 19 14 23 DNAArtificial Sequence Probe 14 aattcatggt ccagaacaac ccg 23 15 20 DNAArtificial Sequence Probe 15 ggttgaccta caagcgccga 20 16 21 DNAArtificial Sequence Probe 16 gggttgacct acaagcgccg a 21 17 23 DNAArtificial Sequence Probe 17 ttgacctaca agcgccgact gtc 23 18 19 DNAArtificial Sequence Probe 18 gttgaccgac aagcgccga 19 19 20 DNAArtificial Sequence Probe 19 ggttgaccga caagcgccga 20 20 23 DNAArtificial Sequence Probe 20 ttgaccgaca agcgccgact gtc 23 21 18 DNAArtificial Sequence Probe 21 cgactgttgg cgctgggg 18 22 19 DNA ArtificialSequence Probe 22 cgactgttgg cgctggggc 19 23 21 DNA Artificial SequenceProbe 23 ggacgtggag gcgatcacac c 21 24 23 DNA Artificial Sequence Probe24 cgatcacacc gcagacgttg atc 23 25 18 DNA Artificial Sequence Probe 25catccggccg gtggtcgc 18 26 17 DNA Artificial Sequence Probe 26 ctggggcccggcggtct 17 27 20 DNA Artificial Sequence Probe 27 cggtctgtca cgtgagcgtg20 28 18 DNA Artificial Sequence Probe 28 catccgtccc gtcgtggc 18 29 19DNA Artificial Sequence Probe 29 catccgtcca gtcgtggcg 19 30 25 DNAArtificial Sequence Primer 30 gagaattcgg tcggcgagct gatcc 25 31 20 DNAArtificial Sequence Primer 31 ggtcggcatg tcgcggatgg 20 32 20 DNAArtificial Sequence Primer 32 gcacgtcgcg gacctccagc 20 33 23 DNAArtificial Sequence Primer 33 cgaagcttga cccgcgctac acc 23 34 329 DNAArtificial Sequence Probe 34 gatccgggtc ggcatgtcgc ggatggagcg ggtggtccgggagcggatga ccacccagga 60 cgtggaggcg atcacaccgc agacgttgat caacatccggccggtggtcg ccgcgatcaa 120 ggagttcttc ggcaccagcc agctgagcca attcatggaccagaacaacc cgctgtcggg 180 gttgacctgc aagcgccgac tgtcggcgct ggggcccggnggtctgtcac gtgagcgtgc 240 cgggctggag gtccgcgacg tgcacccgtc gcactacggncggatgtgcc ctatcgaaac 300 ccctgagggg gccaacatcg ntctttatc 329 35 319 DNAArtificial Sequence Probe 35 tccgggtcgg catgtcccgg atggagtgtg tcgtccgngagcggatgacc anccaggacg 60 tngaggccat cacgccgcag accctgatca acatccgtcccgtcgtggcg gcgatcaagg 120 agttcttcgg naccagccag ttgtcccagt tcatggaccagaacaacccg ctgtcggggc 180 tcacccacaa gcgccgcctg tcggcgntgg gcccgggtggtctgtcccgg gagcgggccg 240 ggctggaggt ccgngacgtg nacccgtccc actacggccggatgtgcccg atcgagaccc 300 cggagggtcc caacatcgg 319 36 324 DNA ArtificialSequence Probe 36 ggatggagcg ctccgtccgc gagcggatga ccacccagga cgtcgaggccatcacgccgc 60 agaccctgat caacatccgt ccagtcgtgg cggcgatcaa ggagttcttcggcaccagcc 120 agctgtccca gttcatggac cagaacaacc cgctgtcggg gctcacccacaagcgccgcc 180 tgtcggcgct gggcccgggt ggtctgtccc gggagcgggc cgggctggaggtccgcgacg 240 tgcacccgtc ccactacggc cggatgtgcc cgatcgagac cccggagggtcccaacatcg 300 gtctgatcgg ctcgctgtcg gtgt 324 37 254 DNA ArtificialSequence Probe 37 cgggtcggca tgtcccgcat ggagcgggtc gtccgcgagc ggatgaccacgcaggacgtc 60 gaggcgatca cgccgcagac cctgatcaac atccggccgg tcgtggccgcgatcaaggag 120 ttcttcggca ccagccagct ctcgcagttc atggaccaga acaacccgntgtcgggcctg 180 acccacaagc gccgcctgtc ggtgctgggc ccggttggtc tgtcccgcgagcgggccggg 240 ttggaggtcc ggag 254 38 16 DNA Artificial Sequence Probe38 gccggtcgtg gccgcg 16 39 19 DNA Artificial Sequence Probe 39gactgtcggc gctggggcc 19 40 18 DNA Artificial Sequence Probe 40gactgtcggc gctggggc 18 41 21 DNA Artificial Sequence Primer 41cggcatgtcg cggatggagc g 21 42 21 DNA Artificial Sequence Primer 42cggctcgctg tcggtgtacg c 21 43 22 DNA Artificial Sequence Probe 43ttgacccaca agcgccgact gt 22 44 19 DNA Artificial Sequence Probe 44gccccagcgc cgacagtcg 19 45 19 DNA Artificial Sequence Probe 45cgactgtcgg cgttggggc 19 46 21 DNA Artificial Sequence Probe 46cagccagccg agccaattca t 21 47 23 DNA Artificial Sequence Probe 47aattcatgta ccagaacaac ccg 23 48 21 DNA Artificial Sequence Probe 48atggaccaga acccgctgtc g 21 49 21 DNA Artificial Sequence Probe 49aacccgctgt tggggttgac c 21 50 23 DNA Artificial Sequence Probe 50ttgacccgca agcgccgact gtc 23 51 23 DNA Artificial Sequence Probe 51ttgaccccca agcgccgact gtc 23 52 23 DNA Artificial Sequence Probe 52ttgacctgca agcgccgact gtc 23 53 19 DNA Artificial Sequence Probe 53cgactgtggg cgctggggc 19 54 19 DNA Artificial Sequence Probe 54actgtgggcg ccggggccc 19 55 18 DNA Artificial Sequence Probe 55agcgccggct ttcggcgc 18 56 243 DNA Artificial Sequence Probe 56ggatggaacg ggtggtccgg gnnnnggatg accactcagg acgtcgaggc gatcacgccg 60agacactgat caacatccgc ccggtggtcg ccgccatcaa ggagttcttc ggcaccagcc 120agctctccca gttcatggac cagaacaacc cgctgtcggg cctcacccac aagcgccggc 180tttcggcgct ggggccgggc ggtctgtccc gggagcgtgc cgggctggag gtccgcgatg 240ctc 243 57 23 DNA Artificial Sequence Probe 57 gacgctgatc aatatccgtc cgg23 58 22 DNA Artificial Sequence Probe 58 cagccagctg tcgcagttca tg 22 5923 DNA Artificial Sequence Probe 59 gttcatggat cagaacaacc ctc 23 60 21DNA Artificial Sequence Probe 60 aaccctctgt cgggcctgac c 21 61 20 DNAArtificial Sequence Probe 61 acccacaagc gccggctgtc 20 62 17 DNAArtificial Sequence Probe 62 ggctgtcggc gctgggc 17 63 22 DNA ArtificialSequence Probe 63 ctgtcgcagt tcatggatca ga 22 64 21 DNA ArtificialSequence Primer 64 ccaaaaccag atccgggtcg g 21 65 20 DNA ArtificialSequence Primer 65 gtccgggagc ggatgaccac 20 66 20 DNA ArtificialSequence Primer 66 gggtgcacgt cgcggacctc 20 67 20 DNA ArtificialSequence Primer 67 gggcacatcc ggccgtagtg 20 68 18 DNA ArtificialSequence Probe 68 atccggccgg tcgtcgcc 18 69 228 DNA Artificial SequenceProbe 69 ggcatttnac ggatggaacg cgtggtccgc gancggatga ccacgcaggacgtcgaggcc 60 atcacgccgc agaccctgat caacatccgg ccggtcgtcg ccgcgatcaaggagttcttc 120 gggaccagcc agctgtcgca gttcatggac cagaacaacc cgctgtcgggtctgacccac 180 aagcgtcgcc tgtcggcgct gggtcccggc ggtctgtccc gtgagcgc 22870 20 DNA Artificial Sequence Probe 70 tacggtcggc gagctgatcc 20 71 19DNA Artificial Sequence Probe 71 tacggcgttt cgatgaacc 19 72 80 DNAArtificial Sequence Probe 72 c agc cag ctg agc caa ttc atg gac cag aacaac ccg ctg tcg ggg ttg 49 Ser Gln Leu Ser Gln Phe Met Asp Gln Asn AsnPro Leu Ser Gly Leu 1 5 10 15 acc cac aag cgc cga ctg tcg gcg ctg ggg c80 Thr His Lys Arg Arg Leu Ser Ala Leu Gly 20 25 73 26 PRT ArtificialSequence Probe 73 Ser Gln Leu Ser Gln Phe Met Asp Gln Asn Asn Pro LeuSer Gly Leu 1 5 10 15 Thr His Lys Arg Arg Leu Ser Ala Leu Gly 20 25

1. Method for the detection of the antibiotic resistance spectrum ofMycobacterium species present in a sample, possibly coupled to theidentification of the Mycobacterium species involved, comprising thesteps of: (i) if need be releasing, isolating or concentrating thepolynucleic acids present in the sample; (ii) if need be amplifying therelevant part of the antibiotic resistance genes present in said samplewith at least one suitable primer pair as described in claims 22 to 24;(iii) hybridizing the polynucleic acids of step (i) or (ii) with atleast one of the rpoB gene probes, as specified in table 2, underappropiate hybridization and wash conditions; (iv) detecting the hybridsformed in step (iii); (v) inferring the Mycobacterium antibioticresistance spectrum, and possibly the Mycobacterium species involved,from the differential hybridization signal(s) obtained in step (iv). 2.Method according to claim 1 for detection of rifampicin (and/orrifabutin) resistance of M. tuberculosis present in a biological sample,comprising the steps of: (i) if need be releasing, isolating orconcentrating the polynucleic acids present in the sample; (ii) if needbe amplifying the relevant part of the rpoB gene with at least onesuitable primer pair as described in claims 22 to 23; (iii) hybridizingthe polynucleic acids of step (i) or (ii) with a set of probes, underappropiate hybridization and wash conditions, with said set comprisingat least one of the following rpoB wild-type or S-probes as specified inTable 2: S1 (SEQ ID NO 1), S11 (SEQ ID NO 2), S2 (SEQ ID NO 3), S3 (SEQID NO 4), S33 (SEQ ID NO 5), S4 (SEQ ID NO 6), S44 (SEQ ID NO 7), S444(SEQ ID NO 43), S4444 (SEQ ID NO 8), S5 (SEQ ID NO 9), S55 (SEQ ID NO10), S555 (SEQ ID NO 39), S5555 (SEQ ID NO 40), S55C (SEQ ID NO 44),555M (SEQ ID NO 45), S6 (SEQ ID NO 11), and/or. S66 (SEQ ID NO 12),

(iv) detecting the hybrids formed in step (iii); (v) inferring therifampicin susceptibility (sensitivity versus resistance) of M.tuberculosis present in the sample from the differential hybridizationsignal(s) obtained in step (iv).
 3. Method according to claim 2, whereinstep (iii) consists of hybridizing with a set of probes comprising atleast 5 or 6 of the specified S-probes of claim 2, wherein said probestaken together cover the mutation region of the rpoB gene.
 4. Methodaccording to claims 2 or 3, wherein step (iii) consists of hybridizingwith a set of probes comprising at least one and preferably all of thefollowing set of S-probes as specified in Table 2: S11 (SEQ ID NO 2), S2(SEQ ID NO 3). S33 (SEQ ID NO 5). S4444 (SEQ ID NO 8). and/or. S55 orS5555 (SEQ ID NO 10 or 40).


5. Method according to any of claims 2 to 4, wherein step (iii) consistsof hybridizing with a set of probes as defined in any of claims 2 to 4,further comprising at least one rpoB silent mutation probe as specifiedin Table 2, such as: SIL-1 (SEQ ID NO 13).
 6. Method according to any ofclaims 2 to 5, wherein step (iii) consists of hybridizing with a set ofprobes as defined in any of claims 2 to 5, with said set furthercomprising at least one rpoB mutation or R-probe, with said probe beingselected from the following list of R-probes as specified in Table 2: R1(SEQ ID NO 46), R2 (SEQ ID NO 14), R2B (SEQ ID NO 47), R2C (SEQ ID NO48). R3 (SEQ ID NO 49). R4A (SEQ ID NO 15). R44A (SEQ ID NO 16), R444A(SEQ ID NO 17), R4B (SEQ ID NO 18), R44B (SEQ ID NO 19), R444B (SEQ IDNO 20). R4C (SEQ ID NO 50), R4D (SEQ ID NO 51), R4E (SEQ ID NO 52), R5(SEQ ID NO 21), R55 (SEQ ID NO 22), R5B (SEQ ID NO 53), and/or. R5C (SEQID NO 54).


7. Method according to claim 6, wherein step (iii) consists ofhybridizing with a set of probes, with said set comprising at least oneof the following R-probes as specified in Table 2: R2 (SEQ ID NO 14),R444A (SEQ ID NO 17), R444B (SEQ ID NO 20). and/or. R55 (SEQ ID NO 22).


8. Method according to claim 2, wherein step (iii) consists ofhybridizing with a set of probes, with said set comprising at least thefollowing S-probes as specified in Table 2: S4444 (SEQ ID NO 8), and.S55 (or 55555) (SEQ ID NO 10 or 40)


9. Method according to claim 2, wherein step (iii) consists ofhybridizing to a set of probes, with said set comprising at least thefollowing S-probes as specified in Table 2: S2 (SEQ ID NO 3). S4444 (SEQID NO 8), and. S55 (or S5555) (SEQ ID NO 10 or 40)


10. Method according to any of claims 8 or 9, wherein step (iii)consists of hybridizing to a set of probes, with said set furthercomprising at least one R-probe chosen from the following list ofR-probes as specified in Table 2: R2 (SEQ ID NO 14), R444A (SEQ ID NO17), R444B (SEQ ID NO 20), and/or. R55 (SEQ ID NO 22).


11. Method according to any of claims 2 to 10 for the simultaneousidentification of Mycobacterium tuberculosis present in a sample and thedetection of its rifampicin (and/or rifabutin) resistance, wherein step(iii) consist of hybridizing to set of probes further comprising atleast one probe specific for Mycobacterium tuberculosis, with said probebeing preferentially contained in the amplified rpoB gene fragment, andwith said probe being preferentially chosen from the following group ofprobes as specified in Table 2: MT-POL-1 (SEQ ID NO 23), MT-POL-2 (SEQID NO 24), MT-POL-3 (SEQ ID NO 25), MT-POL-4 (SEQ ID NO 26). and/or.MT-POL-5 (SEQ ID NO 27).


12. Method according to claim 11, wherein step (iii) consist ofhybridizing to a set of probes comprising the following probe asspecified in Table 2: MT-POL-1 (SEQ ID NO 23).
 13. Method according toany of claims 11 to 12 for the simultaneous identification of M.tuberculosis and mycobacteria other than M. tuberculosis, coupled todetection of rifampicin resistance of M. tuberculosis, wherein step(iii) consists of hybridizing with a set of probes further comprising atleast one of the following species-specific probes as specified in Table2: MP-POL-1 (SEQ ID NO 28), MA-POL-1 (SEQ ID NO 29), MS-POL-1 (SEQ ID NO38), MK-POL-1 (SEQ ID NO 55), MI-POL-1 (SEQ ID NO 68), and/or. ML-POL-1(SEQ ID NO 57),

and/or any M. paratuberculosis specific probe derived from the sequenceof the relevant part of the rpoB gene of M. paratuberculosis (SEQ ID NO35), and/or any M. avium specific probe derived from the sequence of therelevant part of the rpoB gene of M. avium (SEQ ID NO 36), and/or any M.scrofulaceum specific probe derived from the sequence of the relevantpart of the rpoB gene of M. scrofulaceum (SEQ ID NO 37), and/or any M.kansasii specific probe derived from the sequence of the relevant partof the rpoB gene of M. kansasii (SEQ ID NO 56), and/or any MAC-strainspecific probe derived from the sequence of the relevant part of therpoB gene of a MAC-strain (SEQ ID NO 69).
 14. Method according to claim12, wherein step (iii) consists of hybridizing with the following set ofprobes as specified in Table 2: MT-POL-1 (SEQ ID NO 23), S11 (SEQ ID NO2), S2 (SEQ ID NO 3), S33 (SEQ ID NO 5), S4444 (SEQ ID NO 8), S55 orS5555 (SEQ ID NO 10 or 40). R2 (SEQ ID NO 14), R444A (SEQ ID NO 17),R444B (SEQ ID NO 20). and. R55 (SEQ ID NO 22).


15. Method according to any of claims 2 to 14 for the detection ofrifampicin (and/or rifabutin) resistant Mycobacterium tuberculosispresent in a sample using a probe or a set of primers designed tospecifically detect or amplify the new rpoB gene mutations D516G, H526C,H526T and R529Q.
 16. Method according to claim 1 for the detection ofrifampicin (and/or rifabutin) resistance of M. leprae present in abiological sample, comprising the steps of: (i) if need be releasing,isolating or concentrating the polynucleic acids present in the sample;(ii) if need be amplifying the relevant part of the rpoB gene with atleast one suitable primer pair as described in claim 24; (iii)hybridizing the polynucleic acids of step (i) or (ii) with a selectedset of rpoB wild-type probes under appropiate hybridization and washconditions, with said set comprising at least one of the followingprobes as specified in Table 2: ML-S1 (SEQ ID NO 58). ML-S2 (SEQ ID NO59). ML-S3 (SEQ ID NO 60). ML-S4 (SEQ ID NO 61), ML-S5 (SEQ ID NO 62).or, ML-S6 (SEQ ID NO 63).

(iv) detecting the hybrids formed in step (iii); (v) inferring therifampicin susceptibility (sensitivity versus resistance) of M. lepraepresent in the sample from the differential hybridization signal(s)obtained in step (iv).
 17. Method according to claim 16 for thesimultaneous identification of M. leprae and detection of rifampicinresistance of M. leprae, wherein step (iii) consists of hybridizing witha set of probes further comprising the following probe as specified inTable 2: ML-POL-1 (SEQ ID NO 57).
 18. Reversed phase hybridizationmethod comprising any of the probes as defined in any of claims 1 to 17,wherein said oligonucleotide probes are immobilized oil a solid support.19. Reversed phase hybridization method according to claim 18, whereinsaid oligonucleotide probes are immobilized on a membrane strip. 20.Probe as defined in any of claims 1 to
 17. 21. Composition comprisingany of the probes as defined in claims 1 to
 17. 22. Set of primersallowing amplification of the rpoB gene fragment of M. tuberculosis,selected from the following sets of primers as specified in Table 2: P1and P5 (SEQ ID NO 30 and 33), P3 and P4 (SEQ ID NO 31 and 32), P7 and P8(SEQ ID NO 41 and 42), or. P2 and P6, in combination with (P1 and P5) or(P3 and P4) or (P7 and P8).


23. Set of primers according to claim 22, wherein said primers are thefollowing primers as specified in Table 2: P3 and P4 (SEQ ID NO 31 and32).
 24. Set of primers allowing the amplification of the rpoB genefragment in mycobacteria other than M. tuberculosis, composed of a 5′primer selected from MGRPO-1 (SEQ ID NO 64), or, MGRPO-2 (SEQ ID NO 65),and a 3′-primer, selected from: MGRPO-3 (SEQ ID NO 66), or, MGRPO-4 (SEQID NO 67).


25. A kit for inferring the antibiotic resistance spectrum ofmycobacteria present in a biological sample, possibly coupled to theidentification of the mycobacterial species involved, comprising thefollowing components: (i) when appropiate, a means for releasing,isolating or concentrating the polynucleic acids present in the sample;(ii) when appropriate, at least one of the sets of primers according toany of claims 22 to 24; (iii) at least one of the probes as defined inany of claims 1 to 17, possibly fixed to a solid support; (iv) ahybridization buffer, or components necessary for producing said buffer;(v) a wash solution, or components necessary for producing saidsolution; (vi) when appropriate, a means for detecting the hybridsresulting from the preceding hybridization.
 26. Kit according to claim24 for the simultaneous detection of M. tuberculosis and its resistanceto rifampicin, wherein the sets of primers of step (ii) are as definedin claim 22 to 23, and the probes of step (iii) are as defined in any ofclaims 2 to
 15. 27. Kit according to claim 24, for the simultaneousdetection of M. leprae and its resistance to rifampicin, wherein thesets of primers of step (ii) are as defined in claim 24, and the probesof step (iii) are as defined in claims 16 to 17.